Methods and compositions for the treatment of insulin-associated medical conditions

ABSTRACT

A method of increasing insulin content in a pancreatic beta cell is disclosed. The method comprising expressing in the pancreatic beta cell an exogenous polynucleotide encoding at least one microRNA or a precursor thereof, wherein the microRNA is selected from the group consisting of miR-15, miR-16, miR-24, miR-26, miR-27, miR-29, miR-30, miR-129, miR-141, miR-148, miR-182, miR-200, miR-376 and Let-7, thereby increasing the insulin content in the pancreatic beta cell.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates tomicroRNAs, more particularly, but not exclusively, to expression orrepression of same in pancreatic beta cells for modulation of insulinlevels.

In adult beta cells, insulin transcription is tightly regulated by anetwork of transcriptional activators and repressors. A fewtranscriptional repressors of the Insulin gene have been characterized,including, NR×2.2, Insm1 (Insulinoma-Associated 1/IA1), Sox6, Bhlhe22and Crem. Transcription activators have also been characterizedincluding Pdx1, MafA, NeuroD, Pax6 and Isl1 which maintain the beta cellfate and activate insulin transcription in response to elevation inplasma glucose. Thus, a fine balance between these opposingtranscription factors must be kept for effective insulin synthesis.

The recently discovered microRNA (miRNA) family of posttranscriptionalregulators, provide an additional regulatory layer that may play animportant role in the adult endocrine pancreas. miRNAs are important forbeta cell differentiation and specific miRNAs have been proposed toregulate beta cell genes. For example, miR-124a was shown to affect theexpression of FoxA2 and Pdx1, the secretory pathway proteins, SNAP25 andRab3a, as well as the ion channels Kir6.2 and Sur1 [Baroukh, N. et al.(2007) J Biol Chem 282, 19575-19588; El Ouaamari A. et al. (2008)Diabetes 57, 2708-2717].

The involvement of miRNAs in insulin secretion has also beencontemplated. Poy et al. have shown that miR-375 affects insulinsecretion through regulation of myotrophin expression, specifically,they showed that over-expression of miR-375 suppressed glucose-inducedinsulin secretion, and conversely, inhibition of endogenous miR-375function enhanced insulin secretion [Poy M. N. et al. (2004) Nature 432,226-230]. Xia et al. have shown that over-expression of miR375 reducesglucose-induced insulin secretion by down-regulating the expression ofmyotrophin in Nit-1 cells [Xia et al., Mol Biol Rep. (2010) Epub aheadof print]. Roggli E. et al. have shown an increase in miR-21, miR-34aand miR-146a in islets of NOD mice during development of pre-diabeticinsulitis. According to their teachings, overexpression of miR-21 ormiR-146a did not significantly affect insulin content, insulin promoteractivity or pro-insulin mRNA levels, while overexpression of miR-34a ledto a decrease in insulin content and insulin promoter activityaccompanied by a reduction in pro-insulin mRNA level [Roggli E. et al.,Diabetes (2010) 59(4):978-86].

To date, the only knockout model for an islet-enriched miRNA is themouse knockout for miR-375. Genetic loss of miR-375 causes decreasedbeta cell mass due to impaired proliferation. Additionally, miR-375mutants have increased alpha cell numbers, increased plasma level ofglucagon and increased gluconeogenesis in the liver [Poy M. N. et al.(2009) Proc Natl Acad Sci USA]. Thus miR-375 provides an intriguingendocrine phenotype that encourages further evaluation of the role ofmiRNAs in vivo.

miRNAs are subject to extensive processing, including digestion byDrosha in the nucleus and by Dicer1 in the cytoplasm. Deletion of Dicer1in the early pancreatic lineage, using a Pdx1-Cre mouse line resulted ininactivation of the entire miRNA pathway in the early pancreatic bud.This early inactivation of Dicer 1 causes pancreas agenesis, suggestingthat miRNA are indeed important for pancreas organogenesis.

U.S. Application No. US 2009/0131348 describes methods and compositionsof identifying a miRNA expression profile for a medical condition, suchas pancreatic disease, and subsequently disclose methods for diagnosingand treating such as condition, by for example, downregulation of miRNAor by administration of synthetic miRNA molecules.

U.S. Application No. US 2005/227934 describes pancreatic islet microRNAsand methods for inhibiting same. Specifically, U.S. Application No. US2005/227934 teaches anti-pancreatic islet microRNA molecules which arecapable of inhibiting pancreatic islet microRNAs and use of same fortreating diabetes.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a method of increasing an insulin content in apancreatic beta cell, the method comprising expressing in the pancreaticbeta cell an exogenous polynucleotide encoding at least one microRNA ora precursor thereof, wherein the microRNA is selected from the groupconsisting of miR-15, miR-16, miR-24, miR-26, miR-27, miR-29, miR-30,miR-129, miR-141, miR-148, miR-182, miR-200, miR-376 and Let-7, therebyincreasing the insulin content in the pancreatic beta cell.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a condition associated with aninsulin deficiency in a subject in need thereof, the method comprisingadministering to the subject an exogenous polynucleotide encoding atleast one microRNA or a precursor thereof, wherein the microRNA isselected from the group consisting of miR-15, miR-16, miR-24, miR-26,miR-27, miR-29, miR-30, miR-129, miR-141, miR-148, miR-182, miR-200,miR-376 and Let-7, thereby treating the condition associated with aninsulin deficiency.

According to an aspect of some embodiments of the present inventionthere is provided a polynucleotide encoding at least one microRNA or aprecursor thereof, wherein the microRNA is selected from the groupconsisting of miR-15, miR-16, miR-24, miR-26, miR-27, miR-29, miR-30,miR-129, miR-141, miR-148, miR-182, miR-200, miR-376 and Let-7 fortreating a condition associated with an insulin deficiency.

According to an aspect of some embodiments of the present inventionthere is provided a nucleic acid construct comprising a nucleic acidsequence encoding a microRNA or a precursor thereof the nucleic acidsequence being operably linked to a pancreatic beta cell specificpromoter.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising the nucleicacid construct of claim 6 or 7 and a pharmaceutically acceptablecarrier.

According to an aspect of some embodiments of the present inventionthere is provided an isolated pancreatic beta cell comprising thenucleic acid construct of claim 6 or 7.

According to an aspect of some embodiments of the present inventionthere is provided a method of decreasing an insulin content in apancreatic beta cell of a subject in need thereof, the method comprisingadministering to the subject an agent capable of downregulatingexpression of at least one microRNA, wherein the microRNA is selectedfrom the group consisting of miR-15, miR-16, miR-24, miR-26, miR-27,miR-29, miR-30, miR-129, miR-141, miR-148, miR-182, miR-200, miR-376 andLet-7, thereby decreasing the insulin content in the pancreatic betacell.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a condition associated withelevated insulin levels in a subject in need thereof, the methodcomprising administering to the subject an agent capable ofdownregulating expression of at least one microRNA in a pancreatic betacell of the subject, wherein the microRNA is selected from the groupconsisting of miR-15, miR-16, miR-24, miR-26, miR-27, miR-29, miR-30,miR-129, miR-141, miR-148, miR-182, miR-200, miR-376 and Let-7, therebytreating the condition associated with elevated insulin levels.

According to some embodiments of the invention, the polynucleotide isoperably linked to a cis acting regulatory element active in pancreaticbeta cell.

According to some embodiments of the invention, the condition associatedwith an insulin deficiency comprises diabetes mellitus.

According to some embodiments of the invention, the microRNA is selectedfrom the group consisting of miR-15, miR-16, miR-24, miR-26, miR-27,miR-29, miR-30, miR-129, miR-141, miR-148, miR-182, miR-200, miR-376 andLet-7.

According to some embodiments of the invention, the isolated pancreaticbeta cell of claim 9 for the treatment of diabetes.

According to some embodiments of the invention, the agent capable ofdownregulating expression of at least one microRNA comprises an enzyme.

According to some embodiments of the invention, the enzyme comprisesDicer1.

According to some embodiments of the invention, the enzyme is selectedfrom the group consisting of Drosha, Dicer1, TUT4, DGCR8, exportin 5,Argonaute1, Argonaute2, Argonaute3, Argonaute4, TRBP, smad4 and Ran.

According to some embodiments of the invention, the subject is a humansubject.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A-C depict tamoxifen treatment-induced deletion of Dicer1 inmutant mice causes a reduction in Dicer1 and in miRNA levels. FIG. 1Ashows Dicer disruption provoked by deletion of a floxed Dicer allelewith the use of a tamoxifen-inducible Cre recombinase protein under thecontrol of the pancreas specific rat insulin promoter (described indetail in the materials and experimental procedures sectionhereinbelow). Concomitant with Dicer deletion, Cre-mediatedrecombination activated EGFP expression by removal of a stop cassettethat prevented its expression. qRT-PCR preformed on whole islets fromcontrol and βDc^(null) mice at 3 weeks post-induction showed a reductionin Dicer1 mRNA (FIG. 1B) and in two islet enriched miRNAs miR-7 andmiR-375 (FIG. 1C).

FIGS. 2A-I depict hyperglycemia and impaired glucose tolerance inβDc^(null) mice due to decreased insulin levels. FIGS. 2A-B show fed(FIG. 2A) and fasting (FIG. 2B) plasma glucose levels in control andβDc^(null) mice 2 and 3 weeks after tamoxifen treatment; FIGS. 2C-D showa glucose tolerance test performed 2 weeks (FIG. 2C) and 3 weeks (FIG.2D) after tamoxifen treatment; FIG. 2E shows pancreatic insulin contentthree weeks after tamoxifen treatment; and FIGS. 2F-I show micrographsof insulin immunohistochemical staining of pancreas sections fromcontrol (FIGS. 2F-G) and βDc^(null) mice (FIG. 2H-I) 3 weeks aftertamoxifen treatment. Panels 2G and 21 are a magnified view of 2F and 2H.Scale bar represents 20 μm.

FIG. 2J depict similar beta cell mass of βDc^(null) animals compared tocontrol mice. Analysis of pancreatic tissue that was isolated fromage-matched animals at 3 weeks post Tamoxifen treatment. The mean (±SEM)islet cell mass of control (n=2) and βDc^(null) animals (n=3) tissueswas calculated by morphometry.

FIGS. 3A-K depict ceased insulin synthesis in βDc^(null) beta cells.Control (FIGS. 3A-3D) and βDc^(null) (FIGS. 3E-H) mice 3 weeks aftertamoxifen treatment were stained for insulin (FIGS. 3B and 3F) and forGFP expression that marks the recombined cells within the islet (FIGS.3A and 3E). The mosaic expression of GFP reflects incomplete inductionby Tamoxifen. In the merged view (FIGS. 3C, 3D, 3G and 3H),co-localization of insulin and GFP staining yields a yellow color incontrol (FIGS. 3C and 3D) but not in βDc^(null) mice (FIGS. 3G and 3H).Scale bar represents 20 μm. FIGS. 3I-K show a reduction in the mRNAlevels of insulin 1 (FIG. 31), insulin 2 (FIG. 3J) and Cre (FIG. 3K) inβDc^(null) mice relative to control by qPCR analysis on isolated islets.Values shown are mean±SEM. * p <0.05, **p <0.01.

FIGS. 4A-L depict maintained beta cell characteristics and no acquiredalternate endocrine markers in βDc^(null) beta cells. Immunofluorescentstaining of the endocrine marker synaptophysin (marked by red, FIGS. 4Aand 4B) and hormone markers—a mixture of antibodies raised againstglucagon, Gehrlin, pancreatic polypeptide and somatostatin (marked byred, FIGS. 4C and 4D)—was indistinguishable between control (FIGS. 4Aand 4C) and βDc^(null) mutants (FIGS. 4B and 4D). Similarly,characteristic beta-cell transcription factors (marked by red) Pdx1(FIGS. 4E and 4F) MafA (FIGS. 4G and 4H) NRx6.1 (FIGS. 4I and 4J) Pax6(FIGS. 4K and 4L) were co-expressed with GFP (marked by green) both incontrols (FIGS. 4E, 4G, 4I and 4K), and in βDc^(null) mutants (FIGS. 4F,4H, 4J and 4L). Scale bar represents 20 μm. Yellow indicatescolocalization.

FIGS. 5A-B depict upregulated expression of transcriptional repressorsof insulin in βDc^(null) islets. qPCR study of RNA isolated from controland βDc^(null) islets, revealed no significant change in the expressionlevels of the transcriptional activators Pdx1, Nkx2.2, Nkx6.1, MafA orNeuroD1 (FIG. 5A, right). However, the levels of some of thetranscriptional repressors examined—Stx3, Hes1, Crem, Insm1, Sox6, Tle4and Bhlhe22—showed a significant increase (FIG. 5A, left).Over-expression of the repressors Sox6 and Bhlhe22 in the HIT beta-cellline caused a reduction in insulin expression which was measured by thereduction in luciferase activity driven by the RIP1 promoter. **p <0.01.

FIGS. 6A-C depict repressed Sox6 or Bhlhe22 expression by a few miRNAs.FIG. 6A depicts a schematic representation of the constructs containingluciferase (luc) sequences fused to the 3′UTR sequences of Sox6 andBhlhe22 (SEQ ID NOs: 42 and 41, respectively). Vertical bars mark thelocation of predicted target sites of the miRNAs that affect luciferaseexpression. These constructs were co-transfected with various miRNAexpression constructs predicted to target these UTRs. The expression ofonly a few of the miRNAs causes a reduction in luciferase activitydriven by the Bhlhe22 (FIG. 6B) or Sox6 UTR (FIG. 6C). The barsrepresent % luciferase activity relative to a non-targeting (nt) controlmiRNA. Values shown are mean±SEM. * p <0.05, **p <0.01.

FIGS. 7A-F depict βDc^(null) beta cells maintain normal PC1/3expression. Immunostaining of PC1/3 (FIGS. 7B and 7E) and GFP expression(FIGS. 7A and 7D) showed their co-localization in the merged picture(FIGS. 7C and 7F, yellow) in both βDc^(null) (FIGS. 7D-F) and control(FIGS. 7A-C) animals.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates tomicroRNAs, more particularly, but not exclusively, to expression orrepression of same in pancreatic beta cells for modulation of insulinlevels.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways. Also,it is to be understood that the phraseology and terminology employedherein is for the purpose of description and should not be regarded aslimiting.

While reducing the present invention to practice, the present inventorshave uncovered that insulin levels may be modified in pancreatic betacells by modulation of microRNAs.

As is illustrated hereinbelow and in the Examples section which followsthe present inventors have uncovered that down-regulation of microRNAsleads to a significant decrease in insulin levels in pancreatic betacells. Specifically, the present inventors have shown that depletion inthe microRNA processing enzyme, Dicer1, in adult beta cells causes overtdiabetes in mice (βDc^(null) mice, see Example 1). The diabetes in thesemice was not caused by loss of beta cell mass but rather by a decreasein insulin levels in the pancreatic beta cells (see Example 2). Furtherresearch showed a significant loss of transcriptional activity in theinsulin promoter in the Dicer1 mutant cells (see Example 3) which wasaccompanied by an abnormal upregulation in transcriptional repressors(e.g. Sox6 and Bhlhe22, see Example 5). Moreover, the present inventorshave uncovered that specific microRNAs (e.g. miR-24, miR-129, miR-15/16,miR-26, miR-27, miR182 and miR-375) are capable of binding andrepressing the transcriptional repressors (see Example 6), thus,allowing reactivation of insulin transcription. The present inventorsconclude that modulation of microRNAs may be used to activate or repressthe Insulin 1 and Insulin 2 genes in pancreatic beta cells.

Thus, according to one aspect of the present invention there is provideda method of increasing insulin content in a pancreatic beta cell, themethod comprising expressing in the pancreatic beta cell an exogenouspolynucleotide encoding at least one microRNA or a precursor thereofthereby increasing the insulin content in the pancreatic beta cell.

According to a specific embodiment, the microRNA of the presentinvention may comprise miR-15, miR-16, miR-24, miR-26a,b, miR-27a,b,miR-29, miR-30a,b,c,d,e, miR-129, miR-141, miR-148a,b, miR-182,miR-200a,b,c, miR-376 and/or Let7a,b,c,d,e,f,g,i.

The phrase “pancreatic beta cell” as used herein refers to pancreaticislet endocrine cells which are capable of secreting insulin in responseto elevated glucose concentrations and express typical beta cellmarkers. Examples of beta cell markers include, but are not limited to,insulin, pdx, Hnf313, PC1/3, Beta2, Nkx2.2, GLUT2 and PC2.

According to the present teachings, the pancreatic beta cell may be partof a tissue (e.g. within a body) or may comprise isolated cells. Theisolated pancreatic beta cells may be of homogeneous or heterogeneousnature. Thus, for example, the pancreatic beta cells may be comprised inisolated pancreatic islets. Islet cells are typically comprised of thefollowing: 1) beta cells that produce insulin; 2) alpha cells thatproduce glucagon; 3) delta cells (or D cells) that produce somatostatin;and/or F cells that produce pancreatic polypeptide. The polypeptidehormones (insulin, glucagon, somatostatin and pancreatic polypeptide)inside these cells are stored in secretary vesicles in the form ofsecretory granules.

Methods of isolating islets are well known in the art. For example,islets may be isolated from pancreatic tissue using collagenase andficoll gradients. An exemplary method is described in U.S. PatentApplication No. 20080014182, incorporated herein by reference.

It will be appreciated that depending on the intended use of the betacells (explained in further detail, hereinbelow), the pancreatic betacells may be isolated from the islets (e.g. by FACS sorting), may bedispersed into a single cell suspension (e.g. by the addition of trypsinor by trituration) and/or may be cultured (e.g. in cell medium such asCMRL-1066, available from e.g. Cellgro, Mediatech, Inc.). Thus,expressing microRNAs in pancreatic beta cells may be effected in vivo,ex vivo or in vitro.

As used herein, the phrase “insulin content” refers to the amount ofmature insulin inside a pancreatic beta cell.

According to the present teachings, microRNA expression in a pancreaticbeta cell results in an increase in insulin content as a result of anincrease in insulin transcription and/or post transcriptional controland/or increase in insulin translation and/or post-translationalcontrol. The increase in insulin content in the pancreatic beta cellaccording to the present teachings may also result from enhanced insulinstorage and/or retarding insulin breakdown.

Measurement of insulin content is well known in the art. An exemplarymethod is extraction of cellular insulin with 3 M acetic acid. Theamount of mature insulin extracted from the pancreatic beta cell may bedetermined using an ELISA kit commercially available from e.g. Mercodia,Uppsala, Sweden.

As used herein, the term “microRNA or a precursor thereof” refers to themicroRNA (miRNA) molecules acting as post-transcriptional regulators.MicroRNAs are typically processed from pre-miR (pre-microRNAprecursors). Pre-miRs are a set of precursor miRNA molecules transcribedby RNA polymerase III that are efficiently processed into functionalmiRNAs, e.g., upon transfection into cultured cells. A Pre-miR can beused to elicit specific miRNA activity in cell types that do notnormally express this miRNA, thus addressing the function of its targetby down regulating its expression in a “gain of (miRNA) function”experiment. Pre-miR designs exist to all of the known miRNAs listed inthe miRNA Registry and can be readily designed for any research.

It will be appreciated that the microRNAs of the present teachings maybind, attach, regulate, process, interfere, augment, stabilize and/ordestabilize any microRNA target. Such a target can be any molecule,including, but not limited to, DNA molecules, RNA molecules andpolypeptides, such as but not limited to, transcriptional repressors(e.g. Sox6, Bhlhe22, Crem, Insm1 and Tle4).

It will be appreciated that the microRNAs of the present invention arepart of, involved in and/or are associated with an insulin transcriptionpathway. Such a microRNA can be identified via various databasesincluding for example the micro-RNA registry(http://wwwdotsangerdotacdotuk/Software/Rfam/mirna/indexdotshtml).

Thus, according to the present teachings, the insulin content within apancreatic beta cell may be increased (e.g. upregulated) by expressionof a microRNA polynucleotide.

The term “polynucleotide” refers to a single-stranded or double-strandedoligomer or polymer of ribonucleic acid (RNA), deoxyribonucleic acid(DNA) or mimetics thereof. This term includes polynucleotides and/oroligonucleotides derived from naturally occurring nucleic acidsmolecules (e.g., RNA or DNA), synthetic polynucleotide and/oroligonucleotide molecules composed of naturally occurring bases, sugars,and covalent internucleoside linkages (e.g., backbone), as well assynthetic polynucleotides and/or oligonucleotides having non-naturallyoccurring portions, which function similarly to respective naturallyoccurring portions.

The length of the polynucleotide of the present invention is optionallyof 100 nucleotides or less, optionally of 90 nucleotides or less,optionally 80 nucleotides or less, optionally 70 nucleotides or less,optionally 60 nucleotides or less, optionally 50 nucleotides or less,optionally 40 nucleotides or less, optionally 30 nucleotides or less,e.g., 29 nucleotides, 28 nucleotides, 27 nucleotides, 26 nucleotides, 25nucleotides, 24 nucleotides, 23 nucleotides, 22 nucleotides, 21nucleotides, 20 nucleotides, 19 nucleotides, 18 nucleotides, 17nucleotides, 16 nucleotides, 15 nucleotides, optionally between 12 and24 nucleotides, optionally between 5-15, optionally, between 5-25, mostpreferably, about 20-25 nucleotides.

The polynucleotides (including oligonucleotides) designed according tothe teachings of the present invention can be generated according to anyoligonucleotide synthesis method known in the art, including bothenzymatic syntheses or solid-phase syntheses. Equipment and reagents forexecuting solid-phase synthesis are commercially available from, forexample, Applied Biosystems. Any other means for such synthesis may alsobe employed; the actual synthesis of the oligonucleotides is well withinthe capabilities of one skilled in the art and can be accomplished viaestablished methodologies as detailed in, for example: Sambrook, J. andRussell, D. W. (2001), “Molecular Cloning: A Laboratory Manual”;Ausubel, R. M. et al., eds. (1994, 1989), “Current Protocols inMolecular Biology,” Volumes I-III, John Wiley & Sons, Baltimore, Md.;Perbal, B. (1988), “A Practical Guide to Molecular Cloning,” John Wiley& Sons, New York; and Gait, M. J., ed. (1984), “OligonucleotideSynthesis”; utilizing solid-phase chemistry, e.g. cyanoethylphosphoramidite followed by deprotection, desalting, and purificationby, for example, an automated trityl-on method or HPLC.

It will be appreciated that a polynucleotide comprising an RNA moleculecan be also generated using an expression vector as is further describedhereinbelow.

Preferably, the polynucleotide of the present invention is a modifiedpolynucleotide. Polynucleotides can be modified using various methodsknown in the art.

For example, the oligonucleotides or polynucleotides of the presentinvention may comprise heterocylic nucleosides consisting of purines andthe pyrimidines bases, bonded in a 3′-to-5′ phosphodiester linkage.

Preferably used oligonucleotides or polynucleotides are those modifiedeither in backbone, internucleoside linkages, or bases, as is broadlydescribed hereinunder.

Specific examples of preferred oligonucleotides or polynucleotidesuseful according to this aspect of the present invention includeoligonucleotides or polynucleotides containing modified backbones ornon-natural internucleoside linkages. Oligonucleotides orpolynucleotides having modified backbones include those that retain aphosphorus atom in the backbone, as disclosed in U.S. Pat. Nos.4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423;5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939;5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821;5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

Preferred modified oligonucleotide or polynucleotide backbones include,for example: phosphorothioates; chiral phosphorothioates;phosphorodithioates; phosphotriesters; aminoalkyl phosphotriesters;methyl and other alkyl phosphonates, including 3′-alkylene phosphonatesand chiral phosphonates; phosphinates; phosphoramidates, including3′-amino phosphoramidate and aminoalkylphosphoramidates;thionophosphoramidates; thionoalkylphosphonates;thionoalkylphosphotriesters; and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogues of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts, and free acidforms of the above modifications can also be used.

Alternatively, modified oligonucleotide or polynucleotide backbones thatdo not include a phosphorus atom therein have backbones that are formedby short-chain alkyl or cycloalkyl internucleoside linkages, mixedheteroatom and alkyl or cycloalkyl internucleoside linkages, or one ormore short-chain heteroatomic or heterocyclic internucleoside linkages.These include those having morpholino linkages (formed in part from thesugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide,and sulfone backbones; formacetyl and thioformacetyl backbones;methylene formacetyl and thioformacetyl backbones; alkene-containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts, as disclosed inU.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141;5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677;5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240;5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070;5,663,312; 5,633,360; 5,677,437; and 5,677,439.

Other oligonucleotides or polynucleotides which may be used according tothe present invention are those modified in both sugar and theinternucleoside linkage, i.e., the backbone of the nucleotide units isreplaced with novel groups. The base units are maintained forcomplementation with the appropriate polynucleotide target. An exampleof such an oligonucleotide mimetic includes a peptide nucleic acid(PNA). A PNA oligonucleotide refers to an oligonucleotide where thesugar-backbone is replaced with an amide-containing backbone, inparticular an aminoethylglycine backbone. The bases are retained and arebound directly or indirectly to aza-nitrogen atoms of the amide portionof the backbone. United States patents that teach the preparation of PNAcompounds include, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262; each of which is herein incorporated byreference. Other backbone modifications which may be used in the presentinvention are disclosed in U.S. Pat. No. 6,303,374.

Oligonucleotides or polynucleotides of the present invention may alsoinclude base modifications or substitutions. As used herein,“unmodified” or “natural” bases include the purine bases adenine (A) andguanine (G) and the pyrimidine bases thymine (T), cytosine (C), anduracil (U). “Modified” bases include but are not limited to othersynthetic and natural bases, such as: 5-methylcytosine (5-me-C);5-hydroxymethyl cytosine; xanthine; hypoxanthine; 2-aminoadenine;6-methyl and other alkyl derivatives of adenine and guanine; 2-propyland other alkyl derivatives of adenine and guanine; 2-thiouracil,2-thiothymine, and 2-thiocytosine; 5-halouracil and cytosine; 5-propynyluracil and cytosine; 6-azo uracil, cytosine, and thymine; 5-uracil(pseudouracil); 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl, and other 8-substituted adenines and guanines; 5-halo,particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracilsand cytosines; 7-methylguanine and 7-methyladenine; 8-azaguanine and8-azaadenine; 7-deazaguanine and 7-deazaadenine; and 3-deazaguanine and3-deazaadenine. Additional modified bases include those disclosed in:U.S. Pat. No. 3,687,808; Kroschwitz, J. I., ed. (1990), “The ConciseEncyclopedia Of Polymer Science And Engineering,” pages 858-859, JohnWiley & Sons; Englisch et al. (1991), “Angewandte Chemie,” InternationalEdition, 30, 613; and Sanghvi, Y. S., “Antisense Research andApplications,” Chapter 15, pages 289-302, S. T. Crooke and B. Lebleu,eds., CRC Press, 1993. Such modified bases are particularly useful forincreasing the binding affinity of the oligomeric compounds of theinvention. These include 5-substituted pyrimidines, 6-azapyrimidines,and N-2, N-6, and O-6-substituted purines, including2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S. et al. (1993),“Antisense Research and Applications,” pages 276-278, CRC Press, BocaRaton), and are presently preferred base substitutions, even moreparticularly when combined with 2′-O-methoxyethyl sugar modifications.

According to a specific embodiment, upregulating the function of themiRNA of the present invention is effected using a polynucleotide havinga nucleic acid sequence as set forth in SEQ ID NOs: 43-56 and 59-72 (seeTable 1).

TABLE 1 miRNA polynucleotide sequences miRNA Sequence: miR-30uguaaacauccucgacuggaag (SEQ ID NO: 43)uguaaacauccuugacuggaag (SEQ ID NO: 44)uguaaacauccccgacuggaag (SEQ ID NO: 45) miR-200/uaauacugccggguaaugaugga (SEQ ID NO: 46) 141uaauacugccugguaaugauga (SEQ ID NO: 47)uaacacugucugguaaagaugg (SEQ ID NO: 48)uaacacugucugguaacgaugu (SEQ ID NO: 49) miR-24uggcucaguucagcaggaacag (SEQ ID NO: 50) miR-27uucacaguggcuaaguucugc (SEQ ID NO: 51) Let-7ugagguaguagguuguaugguu (SEQ ID NO: 52)ugagguaguagguuguauaguu (SEQ ID NO: 53)ugagguaguagguugugugguu (SEQ ID NO: 54)ugagguaguagauuguauaguu (SEQ ID NO: 55)ugagguaggagguuguauaguu (SEQ ID NO: 56) miR-375uuuguucguucggcucgcguga (SEQ ID NO: 59) miR-148ucagugcacuacagaacuuugu (SEQ ID NO: 60)ucagugcaucacagaacuuugu (SEQ ID NO: 61) miR-26uucaaguaauccaggauaggcu (SEQ ID NO: 62)uucaaguaauucaggauaggu (SEQ ID NO: 63) miR-182uuuggcaaugguagaacucacaccg (SEQ ID NO: 64) miR-376aacauagaggaaauuucacgu (SEQ ID NO: 65)uggaagacuagugauuuuguugu (SEQ ID NO: 66)uggaagacuugugauuuuguugu (SEQ ID NO: 67)uagcagcacaucaugguuuaca (SEQ ID NO: 68) miR-15uagcagcacguaaauauuggcg (SEQ ID NO: 69) miR-16uagcaccauuugaaaucaguguu (SEQ ID NO: 70) miR-29uagcaccaucugaaaucgguua (SEQ ID NO: 71) miR-129cuuuuugcggucugggcuugc (SEQ ID NO: 72)

As is mentioned hereinabove and is shown in the Examples section whichfollows, micro-RNAs are processed molecules derived from specificprecursors (i.e., pre-miRNA), upregulation of a specific miRNA functioncan be effected using a specific miRNA precursor molecule.

Polynucleotide agents for up-regulating microRNA expression inpancreatic beta cells may be provided to the pancreatic beta cells perse. Such polynucleotide agents are typically administered to thepancreatic beta cells as part of an expression construct. In this case,the polynucleotide agent is ligated in a nucleic acid construct underthe control of a cis-acting regulatory element (e.g. promoter) capableof directing an expression of the microRNA in the pancreatic beta cellsin a constitutive or inducible manner.

Examples of microRNA polynucleotide agents of the present inventioninclude, but are not limited to, miR-15 (e.g. GenBank accession no.NR_(—)029485 RNA), miR-16 (e.g. GenBank accession no. NR_(—)029486),miR-24 (e.g. GenBank accession nos. NR_(—)029496 and NR_(—)029497),miR-26 (e.g. GenBank accession no. NR_(—)029500 and NR_(—)029499),miR-27 (e.g. GenBank accession no. NR_(—)029501 RNA), miR-29 (e.g.GenBank accession no. NR_(—)029503 and NR_(—)029832), miR-129 (e.g.GenBank accession nos. NR_(—)029596 and NR_(—)029697) and miR-182 (e.g.GenBank accession no. NR_(—)029614).

Examples of B cell specific promoters include, but are not limited tothe insulin promoter, Nkx6.1 promoter, Nkx2.2 promoter, IPF-1 promoter,Pdx1 promoter and beta-cell glucokinase (GCK) promoter.

The expression constructs of the present invention may also includeadditional sequences which render it suitable for replication andintegration in eukaryotes (e.g., shuttle vectors). Typical cloningvectors contain transcription and translation initiation sequences(e.g., promoters, enhances) and transcription and translationterminators (e.g., polyadenylation signals). The expression constructsof the present invention can further include an enhancer, which can beadjacent or distant to the promoter sequence and can function in upregulating the transcription therefrom.

Enhancer elements can stimulate transcription up to 1.000-fold fromlinked homologous or heterologous promoters. Enhancers are active whenplaced downstream or upstream from the transcription initiation site.Many enhancer elements derived from viruses have a broad host range andare active in a variety of tissues. For example, the SV40 early geneenhancer is suitable for many cell types. Other enhancer/promotercombinations that are suitable for the present invention include thosederived from polyoma virus or human or murine cytomegalovirus (CMV) andthe long tandem repeats (LTRs) from various retroviruses, such as murineleukemia virus, murine or Rous sarcoma virus, and HIV. See Gluzman, Y.and Shenk, T., eds. (1983). Enhancers and Eukaryotic Gene Expression,Cold Spring Harbor Press, Cold Spring Harbor, N.Y., which isincorporated herein by reference.

Polyadenylation sequences can also be added to the expression constructsof the present invention in order to increase the efficiency ofexpression of the detectable moeity. Two distinct sequence elements arerequired for accurate and efficient polyadenylation: GU- or U-richsequences located downstream from the polyadenylation site and a highlyconserved sequence of six nucleotides, namely AAUAAA, located 11-30nucleotides upstream of the site. Termination and polyadenylationsignals suitable for the present invention include those derived fromSV40.

In addition to the embodiments already described, the expressionconstructs of the present invention may typically contain otherspecialized elements intended to increase the level of expression ofcloned nucleic acids or to facilitate the identification of cells thatcarry the recombinant DNA. For example, a number of animal virusescontain DNA sequences that promote extra-chromosomal replication of theviral genome in permissive cell types. Plasmids bearing these viralreplicons are replicated episomally as long as the appropriate factorsare provided by genes either carried on the plasmid or with the genomeof the host cell.

The expression constructs of the present invention may or may notinclude a eukaryotic replicon. If a eukaryotic replicon is present, thevector is capable of amplification in eukaryotic cells using theappropriate selectable marker. If the construct does not comprise aeukaryotic replicon, no episomal amplification is possible. Instead, therecombinant DNA integrates into the genome of the engineered cell, wherethe promoter directs expression of the desired nucleic acid.

The nucleic acid construct may be introduced into the pancreatic betacells of the present invention using an appropriate gene deliveryvehicle/method (transfection, transduction, etc.) and an appropriateexpression system.

Examples of mammalian expression vectors include, but are not limitedto, pcDNA3, pcDNA3.1(+/−), pGL3, pZeoSV2(+/−), pSecTag2, pDisplay,pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1,pNMT41, and pNMT81, which are available from Invitrogen, pCI which isavailable from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV, which areavailable from Strategene, pTRES which is available from Clontech, andtheir derivatives.

Expression vectors containing regulatory elements from eukaryoticviruses such as retroviruses can be also used. SV40 vectors includepSVT7 and pMT2, for instance. Vectors derived from bovine papillomavirus include pBV-1MTHA, and vectors derived from Epstein-Ban virusinclude pHEBO and p205. Other exemplary vectors include pMSG, pAV009/A⁺,pMTO10/A⁺, pMAMneo-5 and baculovirus pDSVE.

Lipid-based systems may be used for the delivery of these constructsinto the pancreatic beta cells of the present invention. Useful lipidsfor lipid-mediated transfer of the gene are, for example, DOTMA, DOPE,and DC-Chol [Tonkinson et al., Cancer Investigation, 14(1): 54-65(1996)]. Recently, it has been shown that Chitosan can be used todeliver nucleic acids to the intestine cells (Chen J. (2004) World JGastroenterol 10(1):112-116). Other non-lipid based vectors that can beused according to this aspect of the present invention include but arenot limited to polylysine and dendrimers.

The expression construct may also be a virus. Examples of viralconstructs include but are not limited to adenoviral vectors, retroviralvectors, vaccinia viral vectors, adeno-associated viral vectors, polyomaviral vectors, alphaviral vectors, rhabdoviral vectors, lenti viralvectors and herpesviral vectors.

Retroviral vectors represent a class of vectors particularly suitablefor use with the present invention. Defective retroviruses are routinelyused in transfer of genes into mammalian cells (for a review, seeMiller, A. D. (1990). Blood 76, 271). A recombinant retroviruscomprising the polynucleotides of the present invention can beconstructed using well-known molecular techniques. Portions of theretroviral genome can be removed to render the retrovirus replicationmachinery defective, and the replication-deficient retrovirus can thenpackaged into virions, which can be used to infect target cells throughthe use of a helper virus while employing standard techniques. Protocolsfor producing recombinant retroviruses and for infecting cells withviruses in vitro or in vivo can be found in, for example, Ausubel et al.(1994) Current Protocols in Molecular Biology (Greene PublishingAssociates, Inc. & John Wiley & Sons, Inc.). Retroviruses have been usedto introduce a variety of genes into many different cell types,including neuronal cells, epithelial cells, endothelial cells,lymphocytes, myoblasts, hepatocytes, and bone marrow cells.

According to one embodiment, a lentiviral vector, a type of retroviralvector, is used according to the present teachings. Lentiviral vectorsare widely used as vectors due to their ability to integrate into thegenome of non-dividing as well as dividing cells. The viral genome, inthe form of RNA, is reverse-transcribed when the virus enters the cellto produce DNA, which is then inserted into the genome at a randomposition by the viral integrase enzyme. The vector (a provirus) remainsin the genome and is passed on to the progeny of the cell when itdivides. For safety reasons, lentiviral vectors never carry the genesrequired for their replication. To produce a lentivirus, severalplasmids are transfected into a so-called packaging cell line, commonlyHEK 293. One or more plasmids, generally referred to as packagingplasmids, encode the virion proteins, such as the capsid and the reversetranscriptase. Another plasmid contains the genetic material to bedelivered by the vector. It is transcribed to produce thesingle-stranded RNA viral genome and is marked by the presence of the ψ(psi) sequence. This sequence is used to package the genome into thevirion.

A specific example of a suitable lentiviral vector for introducing andexpressing the polynucleotide sequences of the present invention in betacells is the lentivirus pLKO.1 vector.

Another suitable expression vector that may be used according to thisaspect of the present invention is the adenovirus vector. The adenovirusis an extensively studied and routinely used gene transfer vector. Keyadvantages of an adenovirus vector include relatively high transductionefficiency of dividing and quiescent cells, natural tropism to a widerange of epithelial tissues, and easy production of high titers (Russel,W. C. (2000) J Gen Virol 81, 57-63). The adenovirus DNA is transportedto the nucleus, but does not integrate thereinto. Thus the risk ofmutagenesis with adenoviral vectors is minimized, while short-termexpression is particularly suitable for treating cancer cells.Adenoviral vectors used in experimental cancer treatments are describedby Seth et al. (1999). “Adenoviral vectors for cancer gene therapy,” pp.103-120, P. Seth, ed., Adenoviruses: Basic Biology to Gene Therapy,Landes, Austin, Tex.).

A suitable viral expression vector may also be a chimericadenovirus/retrovirus vector combining retroviral and adenoviralcomponents. Such vectors may be more efficient than traditionalexpression vectors for transducing tumor cells (Pan et al. (2002).Cancer Letts 184, 179-188).

Various methods can be used to introduce the expression vectors of thepresent invention into human cells. Such methods are generally describedin, for instance: Sambrook, J. and Russell, D. W. (1989, 1992, 2001),Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory,New York; Ausubel, R. M. et al., eds. (1994, 1989). Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989); Chang, P.L., ed. (1995). Somatic Gene Therapy, CRC Press, Boca Raton, Fla.; Vega,M. A. (1995). Gene Targeting, CRC Press, Boca Raton, Fla.; Rodriguez, R.L. and Denhardt, D. H. (1987). Vectors: A Survey of Molecular CloningVectors and Their Uses, Butterworth-Heinemann, Boston, Mass; and Gilboa,E. et al. (1986). Transfer and expression of cloned genes usingretro-viral vectors. Biotechniques 4(6), 504-512; and include, forexample, stable or transient transfection, lipofection, electroporation,and infection with recombinant viral vectors. In addition, see U.S. Pat.Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

When introducing the expression constructs of the present invention intobeta cells by viral infection the viral dose for infection is at least10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵ orhigher pfu or viral particles.

According to an embodiment of the present invention, there is providedan isolated pancreatic beta cell comprising the nucleic acid constructencoding a microRNA, as detailed above.

As mentioned the pancreatic beta cells of the present invention can betreated in vivo (i.e., inside the organism or the subject) or ex vivo(e.g., in a tissue culture). In case the cells are treated ex vivo, themethod preferably includes a step of administering such cells back tothe individual (ex vivo cell therapy). In vivo and ex vivo therapies arefurther discussed hereinbelow.

As mentioned, the present invention is based on the finding thatdownregulation of microRNA expression levels in pancreatic beta cellsresults in decreased insulin levels. As such, the present invention alsocontemplates decreasing (e.g. downregulating) microRNA levels inpancreatic beta cells in order to decrease insulin content in pancreaticcells.

Downregulation of microRNAs can be effected on the genomic and/or thetranscript level using a variety of molecules which interfere withtranscription and/or translation (e.g., RNA silencing agents, Ribozyme,DNAzyme and antisense), or on the protein level using e.g., antagonists,enzymes that cleave the polypeptide and the like.

Following is a list of agents capable of downregulating expression leveland/or activity of microRNAs.

Downregulation of microRNAs can be achieved by RNA silencing. As usedherein, the phrase “RNA silencing” refers to a group of regulatorymechanisms [e.g. RNA interference (RNAi), transcriptional gene silencing(TGS), post-transcriptional gene silencing (PTGS), quelling,co-suppression, and translational repression] mediated by RNA moleculeswhich result in the inhibition or “silencing” of the expression of acorresponding protein-coding gene. RNA silencing has been observed inmany types of organisms, including plants, animals, and fungi.

As used herein, the term “RNA silencing agent” refers to an RNA which iscapable of inhibiting or “silencing” the expression of a target gene. Incertain embodiments, the RNA silencing agent is capable of preventingcomplete processing (e.g, the full translation and/or expression) of anmRNA molecule through a post-transcriptional silencing mechanism. RNAsilencing agents include noncoding RNA molecules, for example RNAduplexes comprising paired strands, as well as precursor RNAs from whichsuch small non-coding RNAs can be generated. Exemplary RNA silencingagents include dsRNAs such as siRNAs, miRNAs and shRNAs. In oneembodiment, the RNA silencing agent is capable of inducing RNAinterference. In another embodiment, the RNA silencing agent is capableof mediating translational repression.

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs). The corresponding process in plants iscommonly referred to as post-transcriptional gene silencing or RNAsilencing and is also referred to as quelling in fungi. The process ofpost-transcriptional gene silencing is thought to be anevolutionarily-conserved cellular defense mechanism used to prevent theexpression of foreign genes and is commonly shared by diverse flora andphyla. Such protection from foreign gene expression may have evolved inresponse to the production of double-stranded RNAs (dsRNAs) derived fromviral infection or from the random integration of transposon elementsinto a host genome via a cellular response that specifically destroyshomologous single-stranded RNA or viral genomic RNA.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs). Short interfering RNAs derived from diceractivity are typically about 21 to about 23 nucleotides in length andcomprise about 19 base pair duplexes. The RNAi response also features anendonuclease complex, commonly referred to as an RNA-induced silencingcomplex (RISC), which mediates cleavage of single-stranded RNA havingsequence complementary to the antisense strand of the siRNA duplex.Cleavage of the target RNA takes place in the middle of the regioncomplementary to the antisense strand of the siRNA duplex.

Accordingly, the present invention contemplates use of dsRNA todown-regulate protein expression from mRNA.

According to one embodiment, the dsRNA is greater than 30 bp. The use oflong dsRNAs (i.e. dsRNA greater than 30 bp) has been very limited owingto the belief that these longer regions of double stranded RNA willresult in the induction of the interferon and PKR response. However, theuse of long dsRNAs can provide numerous advantages in that the cell canselect the optimal silencing sequence alleviating the need to testnumerous siRNAs; long dsRNAs will allow for silencing libraries to haveless complexity than would be necessary for siRNAs; and, perhaps mostimportantly, long dsRNA could prevent viral escape mutations when usedas therapeutics.

Various studies demonstrate that long dsRNAs can be used to silence geneexpression without inducing the stress response or causing significantoff-target effects—see for example [Strat et al., Nucleic AcidsResearch, 2006, Vol. 34, No. 13 3803-3810; Bhargava A et al. Brain Res.Protoc. 2004; 13:115-125; Diallo M., et al., Oligonucleotides. 2003;13:381-392; Paddison P. J., et al., Proc. Natl. Acad. Sci. USA. 2002;99:1443-1448; Tran N., et al., FEBS Lett. 2004; 573:127-134].

In particular, the present invention also contemplates introduction oflong dsRNA (over 30 base transcripts) for gene silencing in cells wherethe interferon pathway is not activated (e.g. embryonic cells andoocytes) see for example Billy et al., PNAS 2001, Vol 98, pages14428-14433. and Diallo et al, Oligonucleotides, Oct. 1, 2003, 13(5):381-392. doi:10.1089/154545703322617069.

The present invention also contemplates introduction of long dsRNAspecifically designed not to induce the interferon and PKR pathways fordown-regulating gene expression. For example, Shinagwa and Ishii [Genes& Dev. 17 (11): 1340-1345, 2003] have developed a vector, named pDECAP,to express long double-strand RNA from an RNA polymerase II (Pol II)promoter. Because the transcripts from pDECAP lack both the 5′-capstructure and the 3′-poly(A) tail that facilitate ds-RNA export to thecytoplasm, long ds-RNA from pDECAP does not induce the interferonresponse.

Another method of evading the interferon and PKR pathways in mammaliansystems is by introduction of small inhibitory RNAs (siRNAs) either viatransfection or endogenous expression.

The term “siRNA” refers to small inhibitory RNA duplexes (generallybetween 18-30 basepairs) that induce the RNA interference (RNAi)pathway. Typically, siRNAs are chemically synthesized as 21 mers with acentral 19 by duplex region and symmetric 2-base 3′-overhangs on thetermini, although it has been recently described that chemicallysynthesized RNA duplexes of 25-30 base length can have as much as a100-fold increase in potency compared with 21 mers at the same location.The observed increased potency obtained using longer RNAs in triggeringRNAi is theorized to result from providing Dicer with a substrate (27mer) instead of a product (21 mer) and that this improves the rate orefficiency of entry of the siRNA duplex into RISC.

It has been found that position of the 3′-overhang influences potency ofan siRNA and asymmetric duplexes having a 3′-overhang on the antisensestrand are generally more potent than those with the 3′-overhang on thesense strand (Rose et al., 2005). This can be attributed to asymmetricalstrand loading into RISC, as the opposite efficacy patterns are observedwhen targeting the antisense transcript.

The strands of a double-stranded interfering RNA (e.g., an siRNA) may beconnected to form a hairpin or stem-loop structure (e.g., an shRNA).Thus, as mentioned the RNA silencing agent of the present invention mayalso be a short hairpin RNA (shRNA).

The term “shRNA”, as used herein, refers to an RNA agent having astem-loop structure, comprising a first and second region ofcomplementary sequence, the degree of complementarity and orientation ofthe regions being sufficient such that base pairing occurs between theregions, the first and second regions being joined by a loop region, theloop resulting from a lack of base pairing between nucleotides (ornucleotide analogs) within the loop region. The number of nucleotides inthe loop is a number between and including 3 to 23, or 5 to 15, or 7 to13, or 4 to 9, or 9 to 11. Some of the nucleotides in the loop can beinvolved in base-pair interactions with other nucleotides in the loop.Examples of oligonucleotide sequences that can be used to form the loopinclude 5′-UUCAAGAGA-3′ (Brummelkamp, T. R. et al. (2002) Science 296:550) and 5′-UUUGUGUAG-3′ (Castanotto, D. et al. (2002) RNA 8:1454). Itwill be recognized by one of skill in the art that the resulting singlechain oligonucleotide forms a stem-loop or hairpin structure comprisinga double-stranded region capable of interacting with the RNAi machinery.

According to another embodiment the RNA silencing agent may be a miRNA.miRNAs are small RNAs made from genes encoding primary transcripts ofvarious sizes. They have been identified in both animals and plants. Theprimary transcript (termed the “pri-miRNA”) is processed through variousnucleolytic steps to a shorter precursor miRNA, or “pre-miRNA.” Thepre-miRNA is present in a folded form so that the final (mature) miRNAis present in a duplex, the two strands being referred to as the miRNA(the strand that will eventually basepair with the target) The pre-miRNAis a substrate for a form of dicer that removes the miRNA duplex fromthe precursor, after which, similarly to siRNAs, the duplex can be takeninto the RISC complex. It has been demonstrated that miRNAs can betransgenically expressed and be effective through expression of aprecursor form, rather than the entire primary form (Parizotto et al.(2004) Genes & Development 18:2237-2242 and Guo et al. (2005) Plant Cell17:1376-1386).

Unlike, siRNAs, miRNAs bind to transcript sequences with only partialcomplementarity (Zeng et al., 2002, Molec. Cell 9:1327-1333) and represstranslation without affecting steady-state RNA levels (Lee et al., 1993,Cell 75:843-854; Wightman et al., 1993, Cell 75:855-862). Both miRNAsand siRNAs are processed by Dicer and associate with components of theRNA-induced silencing complex (Hutvagner et al., 2001, Science293:834-838; Grishok et al., 2001, Cell 106: 23-34; Ketting et al.,2001, Genes Dev. 15:2654-2659; Williams et al., 2002, Proc. Natl. Acad.Sci. USA 99:6889-6894; Hammond et al., 2001, Science 293:1146-1150;Mourlatos et al., 2002, Genes Dev. 16:720-728). A recent report(Hutvagner et al., 2002, Sciencexpress 297:2056-2060) hypothesizes thatgene regulation through the miRNA pathway versus the siRNA pathway isdetermined solely by the degree of complementarity to the targettranscript. It is speculated that siRNAs with only partial identity tothe mRNA target will function in translational repression, similar to anmiRNA, rather than triggering RNA degradation.

Synthesis of RNA silencing agents suitable for use with the presentinvention can be effected as follows. First, the microRNA mRNA sequenceis scanned downstream of the AUG start codon for AA dinucleotidesequences. Occurrence of each AA and the 3′ adjacent 19 nucleotides isrecorded as potential siRNA target sites. Preferably, siRNA target sitesare selected from the open reading frame, as untranslated regions (UTRs)are richer in regulatory protein binding sites. UTR-binding proteinsand/or translation initiation complexes may interfere with binding ofthe siRNA endonuclease complex [Tuschl ChemBiochem. 2:239-245]. It willbe appreciated though, that siRNAs directed at untranslated regions mayalso be effective, as demonstrated for GAPDH wherein siRNA directed atthe 5′ UTR mediated about 90% decrease in cellular GAPDH mRNA andcompletely abolished protein level(www.ambion.com/techlib/tn/91/912.html).

Second, potential target sites are compared to an appropriate genomicdatabase (e.g., human, mouse, rat etc.) using any sequence alignmentsoftware, such as the BLAST software available from the NCBI server(www.ncbi.nlm.nih.gov/BLAST/). Putative target sites which exhibitsignificant homology to other coding sequences are filtered out.

Qualifying target sequences are selected as template for siRNAsynthesis. Preferred sequences are those including low G/C content asthese have proven to be more effective in mediating gene silencing ascompared to those with G/C content higher than 55%. Several target sitesare preferably selected along the length of the target gene forevaluation. For better evaluation of the selected siRNAs, a negativecontrol is preferably used in conjunction. Negative control siRNApreferably include the same nucleotide composition as the siRNAs butlack significant homology to the genome. Thus, a scrambled nucleotidesequence of the siRNA is preferably used, provided it does not displayany significant homology to any other gene.

It will be appreciated that the RNA silencing agent of the presentinvention need not be limited to those molecules containing only RNA,but further encompasses chemically-modified nucleotides andnon-nucleotides.

In some embodiments, the RNA silencing agent provided herein can befunctionally associated with a cell-penetrating peptide.” As usedherein, a “cell-penetrating peptide” is a peptide that comprises a short(about 12-30 residues) amino acid sequence or functional motif thatconfers the energy-independent (i.e., non-endocytotic) translocationproperties associated with transport of the membrane-permeable complexacross the plasma and/or nuclear membranes of a cell. Thecell-penetrating peptide used in the membrane-permeable complex of thepresent invention preferably comprises at least one non-functionalcysteine residue, which is either free or derivatized to form adisulfide link with a double-stranded ribonucleic acid that has beenmodified for such linkage. Representative amino acid motifs conferringsuch properties are listed in U.S. Pat. No. 6,348,185, the contents ofwhich are expressly incorporated herein by reference. Thecell-penetrating peptides of the present invention preferably include,but are not limited to, penetratin, transportan, pIsl, TAT(48-60), pVEC,MTS, and MAP.

mRNAs to be targeted using RNA silencing agents include, but are notlimited to, those whose expression is correlated with an undesiredphenotypic trait. Exemplary mRNAs that may be targeted are those thatencode truncated proteins i.e. comprise deletions. Accordingly the RNAsilencing agent of the present invention may be targeted to a bridgingregion on either side of the deletion. Introduction of such RNAsilencing agents into a cell would cause a down-regulation of themutated protein while leaving the non-mutated protein unaffected.Exemplary microRNA silencing agents include, but are not limited to,Anti-miR™ miRNA Inhibitors available from Ambion Inc. for inhibition ofmiR-15, miR-16, miR-24, miR-26, miR-27, miR-29, miR-30d, miR-129 andmiR-182 (for more details seehttps://productsdotappliedbiosystemsdotcom/ab/en/US/adirect/ab?cmd=ABAntiPremiRNAKeywordSearch).

Another agent capable of downregulating a microRNA is a DNAzyme moleculecapable of specifically cleaving an mRNA transcript or DNA sequence ofthe microRNA. DNAzymes are single-stranded polynucleotides which arecapable of cleaving both single and double stranded target sequences(Breaker, R. R. and Joyce, G. Chemistry and Biology 1995; 2:655;Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci. USA 1997; 943:4262)A general model (the “10-23” model) for the DNAzyme has been proposed.“10-23” DNAzymes have a catalytic domain of 15 deoxyribonucleotides,flanked by two substrate-recognition domains of seven to ninedeoxyribonucleotides each. This type of DNAzyme can effectively cleaveits substrate RNA at purine:pyrimidine junctions (Santoro, S. W. &Joyce, G. F. Proc. Natl, Acad. Sci. USA 199; for rev of DNAzymes seeKhachigian, L M [Curr Opin Mol Ther 4:119-21 (2002)].

Examples of construction and amplification of synthetic, engineeredDNAzymes recognizing single and double-stranded target cleavage siteshave been disclosed in U.S. Pat. No. 6,326,174 to Joyce et al. DNAzymesof similar design directed against the human Urokinase receptor wererecently observed to inhibit Urokinase receptor expression, andsuccessfully inhibit colon cancer cell metastasis in vivo (Itoh et al,20002, Abstract 409, Ann Meeting Am Soc Gen Ther www.asgt.org). Inanother application, DNAzymes complementary to bcr-ab1 oncogenes weresuccessful in inhibiting the oncogenes expression in leukemia cells, andlessening relapse rates in autologous bone marrow transplant in cases ofCML and ALL.

Downregulation of a microRNA can also be effected by using an antisensepolynucleotide capable of specifically hybridizing with an mRNAtranscript encoding the microRNA.

Design of antisense molecules which can be used to efficientlydownregulate a microRNA must be effected while considering two aspectsimportant to the antisense approach. The first aspect is delivery of theoligonucleotide into the cytoplasm of the appropriate cells, while thesecond aspect is design of an oligonucleotide which specifically bindsthe designated mRNA within cells in a way which inhibits translationthereof.

The prior art teaches of a number of delivery strategies which can beused to efficiently deliver oligonucleotides into a wide variety of celltypes [see, for example, Luft J Mol Med 76: 75-6 (1998); Kronenwett etal. Blood 91: 852-62 (1998); Rajur et al. Bioconjug Chem 8: 935-40(1997); Lavigne et al. Biochem Biophys Res Commun 237: 566-71 (1997) andAoki et al. (1997) Biochem Biophys Res Commun 231: 540-5 (1997)].

In addition, algorithms for identifying those sequences with the highestpredicted binding affinity for their target mRNA based on athermodynamic cycle that accounts for the energetics of structuralalterations in both the target mRNA and the oligonucleotide are alsoavailable [see, for example, Walton et al. Biotechnol Bioeng 65: 1-9(1999)].

Such algorithms have been successfully used to implement an antisenseapproach in cells. For example, the algorithm developed by Walton et al.enabled scientists to successfully design antisense oligonucleotides forrabbit beta-globin (RBG) and mouse tumor necrosis factor-alpha (TNFalpha) transcripts. The same research group has more recently reportedthat the antisense activity of rationally selected oligonucleotidesagainst three model target mRNAs (human lactate dehydrogenase A and Band rat gp130) in cell culture as evaluated by a kinetic PCR techniqueproved effective in almost all cases, including tests against threedifferent targets in two cell types with phosphodiester andphosphorothioate oligonucleotide chemistries.

In addition, several approaches for designing and predicting efficiencyof specific oligonucleotides using an in vitro system were alsopublished (Matveeva et al., Nature Biotechnology 16: 1374-1375 (1998)].

Several clinical trials have demonstrated safety, feasibility andactivity of antisense oligonucleotides. For example, antisenseoligonucleotides suitable for the treatment of cancer have beensuccessfully used [Holmund et al., Curr Opin Mol Ther 1:372-85 (1999)],while treatment of hematological malignancies via antisenseoligonucleotides targeting c-myb gene, p53 and Bcl-2 had enteredclinical trials and had been shown to be tolerated by patients [GerwitzCurr Opin Mol Ther 1:297-306 (1999)].

More recently, antisense-mediated suppression of human heparanase geneexpression has been reported to inhibit pleural dissemination of humancancer cells in a mouse model [Uno et al., Cancer Res 61:7855-60(2001)].

Thus, the current consensus is that recent developments in the field ofantisense technology which, as described above, have led to thegeneration of highly accurate antisense design algorithms and a widevariety of oligonucleotide delivery systems, enable an ordinarilyskilled artisan to design and implement antisense approaches suitablefor downregulating expression of known sequences without having toresort to undue trial and error experimentation.

microRNA antisense agents include, but are not limited to, antisensemolecules which target and inhibit miRNA (e.g. miR-15, miR-16) describedin detail in Cheng A. M. et al., Nucleic Acids Research 200533(4):1290-1297, incorporated herein by reference, and anti-miRNA oligosavailable from e.g. IDT (Integrated DNA Technologies, Inc, Israel) andalso available from Exicon (miRCURY LNA™ microRNA Inhibitors, for moredetails see http://wwwdotexiqondotcom/microrna-knockdown).

Another agent capable of downregulating a microRNA is a ribozymemolecule capable of specifically cleaving an mRNA transcript encoding amicroRNA. Ribozymes are being increasingly used for thesequence-specific inhibition of gene expression by the cleavage of mRNAsencoding proteins of interest [Welch et al., Curr Opin Biotechnol.9:486-96 (1998)]. The possibility of designing ribozymes to cleave anyspecific target RNA has rendered them valuable tools in both basicresearch and therapeutic applications. In the therapeutics area,ribozymes have been exploited to target viral RNAs in infectiousdiseases, dominant oncogenes in cancers and specific somatic mutationsin genetic disorders [Welch et al., Clin Diagn Virol. 10:163-71 (1998)].Most notably, several ribozyme gene therapy protocols for HIV patientsare already in Phase 1 trials. More recently, ribozymes have been usedfor transgenic animal research, gene target validation and pathwayelucidation. Several ribozymes are in various stages of clinical trials.ANGIOZYME was the first chemically synthesized ribozyme to be studied inhuman clinical trials. ANGIOZYME specifically inhibits formation of theVEGF-r (Vascular Endothelial Growth Factor receptor), a key component inthe angiogenesis pathway. Ribozyme Pharmaceuticals, Inc., as well asother firms have demonstrated the importance of anti-angiogenesistherapeutics in animal models. HEPTAZYME, a ribozyme designed toselectively destroy Hepatitis C Virus (HCV) RNA, was found effective indecreasing Hepatitis C viral RNA in cell culture assays (RibozymePharmaceuticals, Incorporated—WEB home page).

Ribozymes specific for targeting microRNAs can be designed as waspreviously described by Suryawanshi, H. et al. [Supplementary Material(ESI) for Molecular BioSystems, The Royal Society of Chemistry 2010,incorporated herein by reference].

An additional method of regulating the expression of a microRNA gene incells is via triplex forming oligonucleotides (TFOs). Recent studieshave shown that TFOs can be designed which can recognize and bind topolypurine/polypirimidine regions in double-stranded helical DNA in asequence-specific manner. These recognition rules are outlined by MaherIII, L. J., et al., Science, 1989; 245:725-730; Moser, H. E., et al.,Science, 1987; 238:645-630; Beal, P. A., et al, Science, 1992;251:1360-1363; Cooney, M., et al., Science, 1988; 241:456-459; andHogan, M. E., et al., EP Publication 375408. Modification of theoligonucleotides, such as the introduction of intercalators and backbonesubstitutions, and optimization of binding conditions (pH and cationconcentration) have aided in overcoming inherent obstacles to TFOactivity such as charge repulsion and instability, and it was recentlyshown that synthetic oligonucleotides can be targeted to specificsequences (for a recent review see Seidman and Glazer, J Clin Invest2003; 112:487-94).

In general, the triplex-forming oligonucleotide has the sequencecorrespondence:

oligo 3′--A G G T duplex 5′--A G C T duplex 3′--T C G A

However, it has been shown that the A-AT and G-GC triplets have thegreatest triple helical stability (Reither and Jeltsch, BMC Biochem,2002, September 12, Epub). The same authors have demonstrated that TFOsdesigned according to the A-AT and G-GC rule do not form non-specifictriplexes, indicating that the triplex formation is indeed sequencespecific.

Thus for any given sequence in the microRNA regulatory region a triplexforming sequence may be devised. Triplex-forming oligonucleotidespreferably are at least 15, more preferably 25, still more preferably 30or more nucleotides in length, up to 50 or 100 bp.

Transfection of cells (for example, via cationic liposomes) with TFOs,and formation of the triple helical structure with the target DNAinduces steric and functional changes, blocking transcription initiationand elongation, allowing the introduction of desired sequence changes inthe endogenous DNA and resulting in the specific downregulation of geneexpression. Examples of such suppression of gene expression in cellstreated with TFOs include knockout of episomal supFG1 and endogenousHPRT genes in mammalian cells (Vasquez et al., Nucl Acids Res. 1999;27:1176-81, and Puri, et al, J Biol Chem, 2001; 276:28991-98), and thesequence- and target specific downregulation of expression of the Ets2transcription factor, important in prostate cancer etiology (Carbone, etal, Nucl Acid Res. 2003; 31:833-43), and the pro-inflammatory ICAM-1gene (Besch et al, J Biol Chem, 2002; 277:32473-79). In addition,Vuyisich and Beal have recently shown that sequence specific TFOs canbind to dsRNA, inhibiting activity of dsRNA-dependent enzymes such asRNA-dependent kinases (Vuyisich and Beal, Nuc. Acids Res 2000;28:2369-74).

Additionally, TFOs designed according to the abovementioned principlescan induce directed mutagenesis capable of effecting DNA repair, thusproviding both downregulation and upregulation of expression ofendogenous genes (Seidman and Glazer, J Clin Invest 2003; 112:487-94).Detailed description of the design, synthesis and administration ofeffective TFOs can be found in U.S. Patent Application Nos. 2003 017068and 2003 0096980 to Froehler et al, and 2002 0128218 and 2002 0123476 toEmanuele et al, and U.S. Pat. No. 5,721,138 to Lawn.

Another agent which can be used along with the present invention todownregulate microRNA is a molecule which prevents microRNA activationor substrate binding.

As is shown in FIG. 1C and is described in Example 1 of the Examplessection which follows, the level of pancreatic beta cell microRNA wassignificantly decreased in Dicer1 depleted cells.

Thus, according to a specific embodiment of the present invention,downregulating the levels of microRNAs in pancreatic beta cells may beeffected by expressing in these cells miRNA digestion enzymes, includingbut not limited to, Drosha (RNASEN, e.g. GenBank accession nos:NP_(—)001093882.1 and NP_(—)037367.3, SEQ ID NOs: 77 and 78,respectively), Dicer1 (e.g. GenBank accession nos: NP_(—)085124.2 andNP_(—)803187.1, SEQ ID NOs: 79 and 80, respectively), DGCR8 (e.g.GenBank accession nos: NP_(—)073564.3 and NP_(—)892029.2, SEQ ID NOs: 81and 82, respectively), exportin 5 (e.g. GenBank accession nos:NP_(—)004866.1 and NP_(—)976035.1, SEQ ID NOs: 83 and 84, respectively),Argonaute1 (EIF2C1, e.g. GenBank accession no: NP_(—)036331.1, SEQ IDNO: 85), Argonaute2 (EIF2C2 e.g. GenBank accession nos:NP_(—)001158095.1 and NP_(—)036286.2, SEQ ID NOs: 86 and 87,respectively), Argonaute3 (EIF2C3 e.g. GenBank accession nos:NP_(—)079128.2 and NP_(—)803171.1, SEQ ID NOs: 88 and 89, respectively),Argonaute4 (EIF2C4 e.g. GenBank accession no: NP_(—)060099.2, SEQ ID NO:90), smad4 (e.g. GenBank accession no: NP_(—)005350.1, SEQ ID NO: 91),Ran (e.g. GenBank accession no: NP_(—)006316.1, SEQ ID NO: 92), TUT4(ZCCHC11 e.g. GenBank accession nos: NP_(—)001009881.1 andNP_(—)056084.1, SEQ ID NOs: 93 and 94, respectively) and/or TRBP/TARBP2(e.g. NP_(—)004169.3, NP_(—)599150.1 and NP_(—)599151.2, SEQ ID NOs:95-97).

Preferably, the polynucleotide of the present invention is a modifiedpolynucleotide. Polynucleotides can be modified using various methodsknown in the art.

For example, the oligonucleotides or polynucleotides of the presentinvention may comprise heterocylic nucleosides consisting of purines andthe pyrimidines bases, bonded in a 3′-to-5′ phosphodiester linkage.

Preferably used oligonucleotides or polynucleotides are those modifiedeither in backbone, internucleoside linkages, or bases, as is broadlydescribed hereinunder.

Specific examples of preferred oligonucleotides or polynucleotidesuseful according to this aspect of the present invention includeoligonucleotides or polynucleotides containing modified backbones ornon-natural internucleoside linkages. Oligonucleotides orpolynucleotides having modified backbones include those that retain aphosphorus atom in the backbone, as disclosed in U.S. Pat. Nos.4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423;5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939;5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821;5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

Preferred modified oligonucleotide or polynucleotide backbones include,for example: phosphorothioates; chiral phosphorothioates;phosphorodithioates; phosphotriesters; aminoalkyl phosphotriesters;methyl and other alkyl phosphonates, including 3′-alkylene phosphonatesand chiral phosphonates; phosphinates; phosphoramidates, including3′-amino phosphoramidate and aminoalkylphosphoramidates;thionophosphoramidates; thionoalkylphosphonates;thionoalkylphosphotriesters; and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogues of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts, and free acidforms of the above modifications can also be used.

Alternatively, modified oligonucleotide or polynucleotide backbones thatdo not include a phosphorus atom therein have backbones that are formedby short-chain alkyl or cycloalkyl internucleoside linkages, mixedheteroatom and alkyl or cycloalkyl internucleoside linkages, or one ormore short-chain heteroatomic or heterocyclic internucleoside linkages.These include those having morpholino linkages (formed in part from thesugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide,and sulfone backbones; formacetyl and thioformacetyl backbones;methylene formacetyl and thioformacetyl backbones; alkene-containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts, as disclosed inU.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141;5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677;5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240;5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070;5,663,312; 5,633,360; 5,677,437; and 5,677,439.

Other oligonucleotides or polynucleotides which may be used according tothe present invention are those modified in both sugar and theinternucleoside linkage, i.e., the backbone of the nucleotide units isreplaced with novel groups. The base units are maintained forcomplementation with the appropriate polynucleotide target. An exampleof such an oligonucleotide mimetic includes a peptide nucleic acid(PNA). A PNA oligonucleotide refers to an oligonucleotide where thesugar-backbone is replaced with an amide-containing backbone, inparticular an aminoethylglycine backbone. The bases are retained and arebound directly or indirectly to aza-nitrogen atoms of the amide portionof the backbone. United States patents that teach the preparation of PNAcompounds include, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262; each of which is herein incorporated byreference. Other backbone modifications which may be used in the presentinvention are disclosed in U.S. Pat. No. 6,303,374.

Oligonucleotides or polynucleotides of the present invention may alsoinclude base modifications or substitutions. As used herein,“unmodified” or “natural” bases include the purine bases adenine (A) andguanine (G) and the pyrimidine bases thymine (T), cytosine (C), anduracil (U). “Modified” bases include but are not limited to othersynthetic and natural bases, such as: 5-methylcytosine (5-me-C);5-hydroxymethyl cytosine; xanthine; hypoxanthine; 2-aminoadenine;6-methyl and other alkyl derivatives of adenine and guanine; 2-propyland other alkyl derivatives of adenine and guanine; 2-thiouracil,2-thiothymine, and 2-thiocytosine; 5-halouracil and cytosine; 5-propynyluracil and cytosine; 6-azo uracil, cytosine, and thymine; 5-uracil(pseudouracil); 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl, and other 8-substituted adenines and guanines; 5-halo,particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracilsand cytosines; 7-methylguanine and 7-methyladenine; 8-azaguanine and8-azaadenine; 7-deazaguanine and 7-deazaadenine; and 3-deazaguanine and3-deazaadenine. Additional modified bases include those disclosed in:U.S. Pat. No. 3,687,808; Kroschwitz, J. I., ed. (1990), “The ConciseEncyclopedia Of Polymer Science And Engineering,” pages 858-859, JohnWiley & Sons; Englisch et al. (1991), “Angewandte Chemie,” InternationalEdition, 30, 613; and Sanghvi, Y. S., “Antisense Research andApplications,” Chapter 15, pages 289-302, S. T. Crooke and B. Lebleu,eds., CRC Press, 1993. Such modified bases are particularly useful forincreasing the binding affinity of the oligomeric compounds of theinvention. These include 5-substituted pyrimidines, 6-azapyrimidines,and N-2, N-6, and O-6-substituted purines, including2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S. et al. (1993),“Antisense Research and Applications,” pages 276-278, CRC Press, BocaRaton), and are presently preferred base substitutions, even moreparticularly when combined with 2′-O-methoxyethyl sugar modifications.

It will be appreciated that the microRNA antisense agents (e.g.anti-miRNA oligos) of the present invention may also comprise chemicalmodifications and/or the addition of moieties, e.g. a cholesterol moiety(e.g. antagomirs). Such molecules have been previously described in e.g.Krützfeldt J. et al., Nature (2005) 438:685-9.

Methods of in vivo and ex vivo (in vitro) expression in eukaryotic cellsare described hereinabove.

As mentioned, the present inventors have shown that microRNA depletionin pancreatic beta cells leads to overt diabetes (see Example 1 in theExamples section which follows). Thus, upregulating microRNA levels maybe beneficial in cases in which a subject endures an insulin deficiencywhile downregulating microRNA expression may be beneficial in cases inwhich a subject endures elevated insulin levels.

Thus, according to another aspect of the present invention there isprovided a method of treating a condition associated with an insulindeficiency in a subject in need thereof.

The term “treating” refers to inhibiting or arresting the development ofa disease, disorder or condition and/or causing the reduction,remission, or regression of a disease, disorder or condition or keepinga disease, disorder or medical condition from occurring in a subject whomay be at risk for the disease disorder or condition, but has not yetbeen diagnosed as having the disease disorder or condition. Those ofskill in the art will understand that various methodologies and assayscan be used to assess the development of a disease, disorder orcondition, and similarly, various methodologies and assays may be usedto assess the reduction, remission or regression of a disease, disorderor condition.

As used herein, the term “subject” refers to an animal, preferably amammal, most preferably a human being, including both young and oldhuman beings of both sexes who suffer from or are predisposed to aninsulin associated disorder.

Diseases or syndromes which are associated with an insulin deficiencyinclude, but are not limited to, type 1 and type 2 diabetes mellitus,metabolic syndrome, type 1 and type 2 diabetes mellitus subtypes,insulin deficiency syndrome, maturity onset diabetes of the young (MODY1-11), and permanent neonatal diabetes mellitus.

According to a specific embodiment of the present invention, the insulindeficiency comprises diabetes.

As used herein “diabetes” refers to a disease resulting either from anabsolute deficiency of insulin (type 1 diabetes) due to a defect in thebiosynthesis or production of insulin, or a relative deficiency ofinsulin in the presence of insulin resistance (type 2 diabetes), i.e.,impaired insulin action, in an organism. The diabetic patient thus hasabsolute or relative insulin deficiency, and displays, among othersymptoms and signs, elevated blood glucose concentration, presence ofglucose in the urine and excessive discharge of urine.

According to the present teachings, in order to treat the insulindeficiency, the subject is administered an agent capable of increasingmiRNA levels e.g. a polynucleotide encoding a microRNA (as detailed infurther detail hereinabove).

According to yet another aspect of the present invention there isprovided a method of treating a condition associated with elevatedinsulin levels.

Diseases or syndromes which are associated with elevated insulin levelsinclude, but are not limited to, hyperinsulinemia, hyperinsulinemichypoglycemia, congenital hyperinsulinism, diffuse hyperinsulinism,insulinomas (i.e. insulin-secreting tumors e.g. islet cell adenoma oradenomatosis or islet cell carcinoma), adult nesidioblastosis,autoimmune insulin syndrome, noninsulinoma pancreatogenous hypoglycemia,reactive hypoglycemia (idiopathic postprandial syndrome), gastricdumping syndrome, drug induced hyperinsulinism and Hypoglycemia due toexogenous (injected) insulin.

According to the present teachings, in order to treat the elevatedinsulin levels, the subject is administered an agent capable ofdecreasing miRNA levels (e.g. an enzyme), such agents are detailed infurther detail hereinabove.

For in vivo therapy, the agent (e.g., a polynucleotide encoding amicroRNA) is administered to the subject as is or as part of apharmaceutical composition (see further detail hereinbelow).

For ex vivo therapy, cells are preferably treated with the agent of thepresent invention (e.g., a polynucleotide encoding a microRNA),following which they are administered to the subject in need thereof.

Administration of the ex vivo treated cells of the present invention canbe effected using any suitable route of introduction, such asintravenous, intraperitoneal, intra-kidney, intra-gastrointestinaltrack, subcutaneous, transcutaneous, intramuscular, intracutaneous,intrathecal, epidural, and rectal. According to presently preferredembodiments, the ex vivo treated cells of the present invention may beintroduced to the individual using intravenous, intra-kidney,intra-gastrointestinal track, and/or intraperitoneal administration.

The pancreatic beta cells of the present invention can be derived fromeither autologous sources or from allogeneic sources such as humancadavers or donors. Since non-autologous cells are likely to induce animmune reaction when administered to the body several approaches havebeen developed to reduce the likelihood of rejection of non-autologouscells. These include either suppressing the recipient immune system orencapsulating the non-autologous cells in immunoisolating, semipermeablemembranes before transplantation.

Encapsulation techniques are generally classified as microencapsulation,involving small spherical vehicles, and macroencapsulation, involvinglarger flat-sheet and hollow-fiber membranes (Uludag, H. et al. (2000).Technology of mammalian cell encapsulation. Adv Drug Deliv Rev 42,29-64).

Methods of preparing microcapsules are known in the art and include forexample those disclosed in: Lu, M. Z. et al. (2000). Cell encapsulationwith alginate and alpha-phenoxycinnamylidene-acetylatedpoly(allylamine). Biotechnol Bioeng 70, 479-483; Chang, T. M. andPrakash, S. (2001) Procedures for microencapsulation of enzymes, cellsand genetically engineered microorganisms. Mol Biotechnol 17, 249-260;and Lu, M. Z., et al. (2000). A novel cell encapsulation method usingphotosensitive poly(allylamine alpha-cyanocinnamylideneacetate). JMicroencapsul 17, 245-521.

For example, microcapsules are prepared using modified collagen in acomplex with a ter-polymer shell of 2-hydroxyethyl methylacrylate(HEMA), methacrylic acid (MAA), and methyl methacrylate (MMA), resultingin a capsule thickness of 2-5 μm. Such microcapsules can be furtherencapsulated with an additional 2-5 μm of ter-polymer shells in order toimpart a negatively charged smooth surface and to minimize plasmaprotein absorption (Chia, S. M. et al. (2002). Multi-layeredmicrocapsules for cell encapsulation. Biomaterials 23, 849-856).

Other microcapsules are based on alginate, a marine polysaccharide(Sambanis, A. (2003). Encapsulated islets in diabetes treatment.Diabetes Thechnol Ther 5, 665-668), or its derivatives. For example,microcapsules can be prepared by the polyelectrolyte complexationbetween the polyanions sodium alginate and sodium cellulose sulphate andthe polycation poly(methylene-co-guanidine) hydrochloride in thepresence of calcium chloride.

It will be appreciated that cell encapsulation is improved when smallercapsules are used. Thus, for instance, the quality control, mechanicalstability, diffusion properties, and in vitro activities of encapsulatedcells improved when the capsule size was reduced from 1 mm to 400 μm(Canaple, L. et al. (2002). Improving cell encapsulation through sizecontrol. J Biomater Sci Polym Ed 13, 783-96). Moreover, nanoporousbiocapsules with well-controlled pore size as small as 7 nm, tailoredsurface chemistries, and precise microarchitectures were found tosuccessfully immunoisolate microenvironments for cells (See: Williams,D. (1999). Small is beautiful: microparticle and nanoparticle technologyin medical devices. Med Device Technol 10, 6-9; and Desai, T. A. (2002).Microfabrication technology for pancreatic cell encapsulation. ExpertOpin Biol Ther 2, 633-646).

Examples of immunosuppressive agents which may be used in conjunctionwith the ex vivo treatment include, but are not limited to,methotrexate, cyclophosphamide, cyclosporine, cyclosporin A,chloroquine, hydroxychloroquine, sulfasalazine (sulphasalazopyrine),gold salts, D-penicillamine, leflunomide, azathioprine, anakinra,infliximab (REMICADE.sup.R), etanercept, TNF.alpha. blockers, abiological agent that targets an inflammatory cytokine, andNon-Steroidal Anti-Inflammatory Drug (NSAIDs). Examples of NSAIDsinclude, but are not limited to acetyl salicylic acid, choline magnesiumsalicylate, diflunisal, magnesium salicylate, salsalate, sodiumsalicylate, diclofenac, etodolac, fenoprofen, flurbiprofen,indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen,nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin,acetaminophen, ibuprofen, Cox-2 inhibitors and tramadol.

The microRNA polynucleotide, microRNA expression vector as well as themicroRNA downregulating agent of the present invention can beadministered to the individual per se or as part of a pharmaceuticalcomposition which also includes a physiologically acceptable carrier.The purpose of a pharmaceutical composition is to facilitateadministration of the active ingredient to an organism.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the agent accountable forthe biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular,intracardiac, e.g., into the right or left ventricular cavity, into thecommon coronary artery, intravenous, inrtaperitoneal, intranasal, orintraocular injections.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuosinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients (upregulating or downregulating agent) effective toprevent, alleviate or ameliorate symptoms of a disorder (e.g., insulinrelated disease) or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. For example, a dose can be formulatedin animal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provide amplelevels of the active ingredient which are sufficient to induce orsuppress the biological effect (minimal effective concentration, MEC).The MEC will vary for each preparation, but can be estimated from invitro data. Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. Detection assayscan be used to determine plasma concentrations. Depending on theseverity and responsiveness of the condition to be treated, dosing canbe of a single or a plurality of administrations, with course oftreatment lasting from several days to several weeks or until cure iseffected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc. The dosage and timing of administration will be responsive to acareful and continuous monitoring of the individual changing condition.

It will be appreciated that animal models exist by which the agents ofthe present invention may be tested prior to human treatment. Forexample, Type I diabetes models include, pancreatectomy in dogs,spontaneous rodent models (e.g. BBDP rats and the NOD mice). Type IIdiabetes models and obese animal models include, db/db (diabetic) mice,Zucker diabetic fatty (ZDF) rats, sand rats (Psammomys obesus) and obeserhesus monkeys.

Regardless of the above, the agents of the present invention areadministered at an amount selected to avoid unwanted side-effectsassociated with elevated concentrations of microRNA.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising a preparation of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition, as is further detailed above.

The agents of the invention can be suitably formulated as pharmaceuticalcompositions which can be suitably packaged as an article ofmanufacture. Such an article of manufacture comprises a label for use intreating an insulin related disease (e.g. diabetes), the packagingmaterial packaging a pharmaceutically effective amount of the microRNAupregulating or downregulating agent.

It will be appreciated that each of the agents or compositions of thepresent invention may be administered in combination with other knowntreatments, including but not limited to, insulin including short-actinginsulin [e.g. lispro (Humalog) or aspart (NovoLog)] and longer actinginsulin [e.g. Neutral Protamine Hagedorn (NPH), Lente, glargine(Lantus), detemir, or ultralente] and oral medication for control ofblood sugar levels e.g. sulfonylurea or biguanide [metforminGlucophage)].

The agents or compositions of the present invention may be administeredprior to, concomitantly with or following administration of the latter.

In order to test treatment efficacy, the subject may be evaluated byphysical examination as well as using any method known in the art, asfor example, by finger stick blood glucose test, fasting plasma glucosetest, oral glucose tolerance test, glycosylated hemoglobin or hemoglobinA1c, body mass index (BMI) and the like.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

General Materials and Experimental Procedures

Mouse Strains

Mice were housed and handled in accordance with protocols approved bythe Institutional Animal Care and Use Committee of WIS. Mouse strainswhich were used herein: Insulin-CreER [previously described in Dor, Y.et al. (2004) Nature 429, 41-46], Dicer1^(floxed) [previously describedin Harfe, B. D. et al. (2005) Proc Natl Acad Sci USA 102, 10898-10903]and Z/EG [previously described in Novak, A. et al. (2000) Genesis 28,147-155]. Transgenic mice were genotyped by PCR on tail DNA extractedusing DirectPCR (Viagen). The following primers were used: Dicer1forward: CCTGACAGTGACGGTCCAAAG (SEQ ID NO: 1), Dicer1 reverse:CATGACTCTTCA-ACTCAAACT (SEQ ID NO: 2), Cre forward: TGCCACGACCAAGTGACAGC(SEQ ID NO: 3), Cre reverse: CCAGGTTACGGATATAGTTCATG (SEQ ID NO: 4), GFPforward: CCTACGGCGTGCAGTGC-TTCAGC (SEQ ID NO: 5) and GFP reverse:CGGCGAGCTGCACGCT-GCGTCCTC (SEQ ID NO: 6). Tamoxifen (Sigma-Aldrich) wasdissolved in corn oil to 20 mg/ml. 5 doses of 8 mg Tamoxifen wereinjected subcutaneously every other day over 10 days to 1-5 month oldRip-Cre-ER;Dicer1 animals.

Pancreas Physiology Assays

Blood glucose was determined using an ‘Acsensia elite’ glucometer.Pancreatic insulin content and serum insulin levels were determinedusing an ultrasensitive rat insulin ELISA kit (Crystal Chem). Glucosetolerance tests were performed by injecting glucose (2 mg/kg)intraperitoneally after overnight fasting.

Pancreatic Histology and Immunohistochemistry

Pancreata were dissected and fixed in 4% paraformaldehyde for 24 h atroom temperature and then processed into paraffin blocks. 3-5 μm thicksections were de-paraffinized, rehydrated and subjected to antigenretrieval in a 2100-Retriever (PickCell Laboratories, The Netherlands).The following primary antibodies were used: guinea pig anti-insulin(1:200; DAKO), rabbit anti-glucagon (1:200; DAKO), rabbitanti-somatostatin (1:200; Zymed), goat anti-GFP (1:100; Abcam), rabbitanti-GFP (1:250; Invitrogen), rabbit anti-Pdx1 (1:5000; Beta CellBiology Consortium), Rabbit anti-Ngn3 (1:300; Santa Cruz), mouseanti-Nkx6.1 (1:100; hybridoma bank), Rabbit anti-mafA (1:100, Bethyl),Rabbit anti-synaptophysin (1:100; DAKO), Rabbit anti-Pax6 (1:300;Chemicon), Goat anti-ghrelin (1:50; Santa Cruz), rabbit anti-activatedcaspase-3 (1:50, cell signaling) and rabbit anti-prohormone convertase1/3 (1:100; Chemicon). Secondary antibodies conjugated to CY2, CY3, orCY5 were all from Jackson Immunoresearch Laboratories (1:100). Insulinimmunohistochemistry was conducted with a secondary antibody conjugatedto biotin (Jackson Immunoresearch Laboratories) followed by incubationwith extravidin-HRP (Sigma) and developed using a DAB substrate kit(Zymed laboratories). Hoechst (Sigma) was used for nuclear counter-stain(1 μg/ml). Fluorescence images were captured using a Zeiss LSM510 LaserScanning/confocal microscope system equipped with a Zeiss camera under amagnification of x 40.

Islet Isolation

Islets were isolated from whole pancreata as was previously described byLacy and Kostianovsky [Lacy, P. E. and Kostianovsky, M. (1967) Diabetes16, 35-39]. Upon laparotomy, the pancreatic duct was identified andretrograde intra-ductal perfusion of 0.166 mg/ml liberase RI or TM(Roche) diluted in Hank's Balanced Salt Solution (Sigma-Aldrich)supplemented with 1.5 mg/ml DNaseI (Roche) was performed through thesphincter of oddi. Islets were hand-picked and frozen in liquidnitrogen.

RNA Quantification by qPCR

Islet RNA was extracted using RNeasy or miRNeasy kit (Qiagen) followingthe manufacturer's instructions. The RNA samples were DNase-I treated oncolumn (Qiagen). cDNA synthesis was carried out using an oligo d(T)primer (Promega) and SuperScript II reverse transcriptase (Invitrogen)following the manufacturer's instructions on 100-500 ng islet RNA. qPCRanalysis of mRNA expression was performed using DyNAmo™ SYBR® Green qPCRkit (Finnzymes) following the manufacturer's instructions. GAPDH andHPRT were used as control reference genes for normalization. All theqPCR reactions were done in a LightCycler® 480 Real-Time PCR System(Roche). All of the primers used for qPCR are detailed in Table 2,below.

TABLE 2 Primers for quantitative real-time PCR of mRNAs GeneForward primer Reverse primer Dicer1 CACGCCTCCTACCACTACAACACCTGGAGAATGCTGCCGTGGGT (SEQ ID NO. 7) (SEQ ID NO. 8) Insulin1CCTGTTGGTGCACTTCCTAC TGCAGTAGTTCTCCAGCTGG (SEQ ID NO. 9) (SEQ ID NO. 10)Insulin2 CGTGGCTTCTTCTACACACCC AGCTCCAGTTGTGCCACTTGT (SEQ ID NO. 11)(SEQ ID NO. 12) Pdx1 TTCCCGAATGGAACCGAGC GTAGGCAGTACGGGTCCTCT(SEQ ID NO. 13) (SEQ ID NO. 14) Nkx2.2 CCGGGCGGAGAAAGGTATGCTGTAGGCGGAAAAGGGGA (SEQ ID NO. 15) (SEQ ID NO. 16) Nkx6.1TCAGTCAAGGTCTGGTTCC CGATTTGTGCTTTTTCAGCA (SEQ ID NO. 17) (SEQ ID NO. 18)MafA AGGAGGAGGTCATCCGACTG CTTCTCGCTCTCCAGAATGTG (SEQ ID NO. 19)(SEQ ID NO. 20) NeouroD1 GACCCAGAAACTGTCTAAAATAGAGAAAGGAGACCAGATCAGGGCTTT CA (SEQ ID NO. 22) (SEQ ID NO. 21) Stx3GAAGGCACGGGATGAAACTAA GGACAGTCCAATAATCAACGCTA (SEQ ID NO. 23)(SEQ ID NO. 24) Hes1 GTCTAAGCCAACTGAAAACACTGATT TGCCTTCTCTAGCTTGGAATGC(SEQ ID NO. 25) (SEQ ID NO. 26) Insm1 CGGCCACCTTCTACAGCTCGGAGGATCACCTGTCTATTCTCA (SEQ ID NO. 27) (SEQ ID NO. 28) CremGCTGAGGCTGATGAAAAACA GCCACACGATTTTCAAGACA (SEQ ID NO. 29)(SEQ ID NO. 30) Sox6 GACAGCGTTCTGTCATCTCAGCAA CGTTCCGGGGTTCCAAAAGTAACA(SEQ ID NO. 31) (SEQ ID NO. 32) Tle4 TTTACAGGCTCAATACCACAGTCTGCACAGATAGCATTTAGTCGTT (SEQ ID NO. 33) (SEQ ID NO. 34) Bhlhe22GGGGAGAGGGAGGTTTAGTG CCCTTTCATCACTTGCCAAT (SEQ ID NO. 35)(SEQ ID NO. 36) HPRT CTGGTTAAGCAGTACAGCCCCAAA TGGCCTGTATCCAACACTTCGAGA(SEQ ID NO. 37) (SEQ ID NO. 38) GAPDH TGGCAAAGTGGAGATTGTTGCCAAGATGGTGATGGGCTTCCCG (SEQ ID NO. 39) (SEQ ID NO. 40)

Cell Culture

HEK-293T and HIT cells (American Type Culture Collection, Manassas, Va.)were maintained in Dullbecco's modified Eagle's medium (DMEM) with 10%fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, and 100U/ml streptomycin. Cells were maintained at 37° C. at a 5% CO₂atmosphere in a humidified incubator. HEK-293T cells were transfectedwith JET-PEI reagent (Poly Plus), while HIT cells were transfected withLipofectamine™ 2000 Reagent (Invitrogen), both according to themanufacturer's instructions.

Luciferase Activity Assays

miR targeting assay: the mouse Bhlhe22 3′ UTR (chr3: 17955876-17957373,SEQ ID NO: 41) and Sox6 3′ UTR (Chr7:122618117-122619352, SEQ ID NO: 42)were subcloned into psiCHECK-2 Vector (Promega) and transfected intoHEK293T cells together with vectors for miRNAs.

Insulin promoter regulation: the overexpression construct: Sox6 [MouseAnnotation: Chromosome 7, NC_(—)000073.5 (122614858 . . . 123174561,complement, SEQ ID NO: 73) corresponding to the human Annotation:Chromosome 11, NC_(—)000011.9 (15991795 . . . 16497918, complement, SEQID NO: 74) MIM: 607257 ID: 55553], Bhlhe22 (pBeta3, amplified andtransferred into pacGFP [Mouse Annotation: Chromosome 3, NC_(—)000069.5(17954402.17957506) ID: 59058, SEQ ID NO: 75, corresponding to the HumanAnnotation: Chromosome 8, NC_(—)000008.10 (65492814.65496186) MIM:613483 ID: 27319, SEQ ID NO: 76]. pcDNA3 (Invitrogen) were transfectedalong with Rat insulin I promoter construct and A20-Renilla luciferaseconstruct For both assays, 48 hrs post-transfection, cells wereharvested and assayed for Firefly and Renilla luciferase activity usingDual luciferase reporter assay system (Promega), according to themanufacturer's instructions.

Histomorphometry

To determine β cell mass, consecutive paraffin sections 5 μm inthickness and 75 μm apart spanning the entire pancreas (approximately 9sections/pancreas) were stained for insulin and counterstained usingHarris' hematoxylin (Sigma). Digital images of sections were obtained ata low magnification (×40) and stitched using NIS-Elements software. Thefraction of β cells was determined by measuring the area of insulinimmuno-reactivity divided by the area of the whole pancreas, determinedby hematoxylin counterstain. The mass of β cells was calculated as theproduct of pancreas weight and the fraction of tissue covered by βcells.

Statistical Analysis

All statistical analyses were performed using the Student's t-testmodule of the Microsoft Excel statistical software. Results aredisplayed as mean±s.e.m of three or more samples/experiments.

Example 1 Disruption of Dicer1 Causes Glucose Intolerance

As Dicer deficiency blocks the output of the entire miRNA repertoire, itprovides an opportunity to assess the overall contribution of thisnetwork to beta cell function in vivo. Inventors have therefore crosseda Dicer1 conditional allele as previously described by Harfe et al.[Harfe, B. D. et al. (2005) Proc Natl Acad Sci USA 102, 10898-10903]onto an inducible CreER transgene that is driven by the rat insulinpromoter (RIP-CreER) previously described by Dor et al. [Dor, Y. et al.(2004) Nature 429, 41-46]. In the resultant RIP-CreER; Dicer1^(flx/flx)mice (called hereafter “βDc^(null)”, for simplicity), miRNA function isintact until injection of tamoxifen (FIG. 1A). Upon Tamoxifen injection,CreER recombinase inactivates the Dicer1 conditional allele and preventsmiRNA processing. Concomitantly, a lacZ/EGFP (Z/EG) reporter transgene[previously described by Novak et al. (2000) Genesis 28, 147-155)],which was crossed into the genetic background of the mouse, is alsosubject to CreER recombinase activation. Thus, cells in whichrecombination occurred lose Dicer1 activity and are labeled with EGFP.In control mice, which are heterozygous for the Dicer1 alleleRIP-CreER;Dicer1^(flx/+) and also harbor the Z/EG reporter transgene(called hereafter control), tamoxifen induction causes loss of only oneDicer allele and activation of EGFP expression.

A quantitative real-time PCR (qPCR) study of Dicer1 mRNA levels inislets of tamoxifen-treated mice revealed a 50% reduction in Dicer1expression. Taking into account the relative abundance of beta cells inislets and the incomplete recombination efficiency, it is likely thatthe level of Dicer1 in the cells that underwent recombination inβDc^(null) animals was close to zero (FIG. 1B). Similarly,representative miRNAs that are characteristic to beta cells (miR7 andmiR375) are down regulated in these cells (FIG. 1C).

βDc^(null) animals developed hyperglycemia at two weeks after tamoxifeninduction as was evident by blood glucose levels in both fasting (FIG.2B) and fed animals (FIG. 2A). The hyperglycemia rapidly deteriorated bythree weeks post-induction (FIGS. 2A-B). Moreover, βDc^(null) animalswere shown to be defective in a glucose tolerance test (GTT), a defectwhich indicates both phases of insulin secretion are affected (FIGS.2C-D). Taken together, these results indicate that deletion of Dicer1 inadult beta cells causes overt diabetes.

Example 2 Disruption of Dicer1 Causes a Decrease in Insulin Protein

In order to determine what causes the severe diabetic phenotype,inventors analyzed insulin content of whole pancreata. Compared tocontrols, βDc^(null) animals showed an 80% decrease in the total insulincontent (FIG. 2E), that likely explains the hyperglycemic state.Surprisingly, morphometric analysis of βDc^(null) pancreata, showed thatthe beta cell mass was not significantly different from that of wildtype mice (FIG. 2J). Furthermore, inventors did not detect apoptosisusing either TUNEL or activated caspase 3 immunostaining (data notshown).

As the diabetic phenotype of βDc^(null) animals is not caused by loss ofbeta cells, inventors next evaluated insulin levels in beta cells at thecellular level. Indeed, while immunohistochemical analysis of insulinexpression shows a uniform and strong expression in controls (FIGS.2F-G), in βDc^(null) animals expression of insulin varies from strong insome cells to non-existent in others (FIGS. 2H-I). Examination of GFPexpression indicated recombination mosaicisim within the islet, whichwas due to the incomplete activation of the tamoxifen-induced CreER.This phenomenon was useful in providing an internal wild type control ata single-cell resolution. Thus, inventors could compare insulin levelsbetween cells within the same islet, that is to say, between GFPpositive cells (in which recombination occurred) and GFP negative cells(in which recombination had not occurred). When such an analysis wascarried out on control pancreata, which are normal for Dicer1conditional allele but undergo EGFP induction, inventors found that EGFPwas co-detected with insulin (FIGS. 3A-D). Notably, in βDc^(null)animals, Dicer-null/EGFP positive cells showed reduced or even totalabsence of insulin expression (FIGS. 3E-H).

Example 3

Insulin transcription is down regulated in the Dicer1 null beta cellsThe observed decrease in insulin expression could result from a defectin any of the steps in insulin production, i.e. transcription,post-transcriptional modification, translation, post-translationalprocessing or degradation. Inventors could not rule out an effect ondegradation. However, posttranslational defects were not very likelysince the antibody used to detect insulin detected both insulin andpro-insulin. Furthermore, the levels of the insulin processing enzymeProhormone convertase 1/3 (PC 1/3) were similar between βDc^(null) andcontrol pancreata (FIGS. 7A-F). Thus, the effect of Dicer1 loss waslikely earlier—loss of transcriptional or posttranscriptionalregulation.

To address a possible effect of miRNA dysregulation on insulintranscription, inventors examined the levels of the two murine insulintranscripts. qPCR performed on isolated islets from mutants showed a 70%decrease in both Insulin1 and Insulin 2 mRNA levels (FIGS. 3I and 3J).Thus, loss of insulin expression originated from changes in insulin mRNAlevels.

While regulation of insulin gene expression was primarily at thetranscriptional level, posttranscriptional regulation could also play arole, i.e. mRNA levels may be downregulated due to exacerbated mRNAdecay. To distinguish between these two possibilities, inventorsexamined the Cre mRNA levels, a transcript whose expression was driven(artificially) by the insulin promoter, yet its stability was regulatedin a separate manner. Inventors found Cre mRNA to be significantlydownregulated (FIG. 3K), suggesting that the insulin promoter wastranscriptionally less active in βDc^(null) beta cells.

Example 4 Dicer1 Mutant Beta Cells Maintain their DifferentiationMarkers

Two alternative scenarios may explain loss of insulin transcription.First, if insulin transcription defines the functional identity ofmature beta cell, the βDc^(null) beta cells might have lost theiridentity, regressing into an earlier state of differentiation or evenassuming a different cell fate altogether. Alternatively, it may be thata regulatory mechanism that specifically affects insulin transcriptionis perturbed in the otherwise-intact beta cells.

Analysis of beta cell identity markers revealed that Dicer 1 mutant betacells (marked by green, FIGS. 4B and 4D) were indistinguishable fromtheir control counterparts (marked by green, FIGS. 4A and 4C). Theymaintained the expression of the secretory vesicle protein,synaptophysin (marked by red, FIGS. 4A and 4B), and did not express anyother hormone marker such as somatostatin, pancreatic polypeptide,glucagon or ghrelin (marked by red, FIGS. 4C and 4D). In order to testwhether the cells assumed a progenitor state inventors performedimmunostaining for the endocrine progenitor marker, Ngn3, but did notfind any expression (data not shown). Therefore, inventors concludedthat Dicer1 mutant beta cells maintained endocrine features and did notexpress alternative fate markers.

In order to further characterize the identity of the Dicer 1 mutant betacells, inventors analyzed the expression of transcription factors thatare typical of mature beta cells, namely Pdx1, MafA, Pax6 and Nkx6.1.Immunostaining of these beta cell markers (marked by red, FIGS. 4E-4L)was similar in GFP positive/Dicer null cells in βDc^(null) mice and GFPpositive cells in the control mice (marked by green, FIGS. 4E-4L).Moreover, quantification of these transcriptional activators by qPCRsuggested that in the βDc^(null) islets, the expression levels of Pdx1,MafA, Nkx2.2 and Nkx6.1 were comparable with the expression in islets ofcontrol littermates (FIG. 5A). Since the nuclear localization of theseproteins appeared to be normal (FIGS. 4E-4L), inventors concluded thatthe repression of the insulin promoter was likely not due to a decreasein the abundance of the transcriptional activators, neither was itrelated to inadequate compartmentalization. Hence, inventors concludedthat βDc^(null) beta cells retained most of their mature molecularidentity markers. In fact, the only change inventors observed in theβDc^(null) beta cells was downregulation of insulin expression.

Example 5 Dicer1 Mutant Beta Cells Upregulate the Expression of a Set ofTranscriptional Repressors

The fact that insulin transcription is downregulated in beta cells thatexpress the chief transcriptional activators of the insulin promoter,could be explained by an abnormal upregulation of transcriptionalrepressors in βDc^(null) beta cells. Inventors therefore went on toquantify the expression levels of known repressors of insulin synthesisby qPCR. Within the nine repressors that were examined, inventors noteda four-fold increase in the mRNA levels of Sox6 and a three-foldincrease in the expression of Bhlhe22. Three other repressors, Crem,Insm1 and Tle4, were upregulated but to a lesser extent, yet in astatistically significant manner (FIG. 5A). Inventors thereforeconsidered Sox6 and Bhlhe22 as prime candidates in controlling thephenotype of βDc^(null) cells downstream of miRNAs, namely therepression of insulin expression.

In order to verify that these repressors can indeed negatively regulateinsulin synthesis, inventors next utilized a beta cell culture system.When Sox6 and Bhlhe22 were overexpressed along with a constructharboring luciferase under the control of the rat insulin promoter incultured HIT cells, inventors were able to recapitulate the reporteddownregulation of insulin expression. Taken together, these resultsindicated that the upregulation of Sox6 and Bhlhe22 reduced insulintranscription (FIG. 5B).

Example 6 A Few miRNAs can Regulate Sox6 and Bhlhe22

As Dicer 1 deficiency blocks the output of the entire miRNA repertoire,inventors did not know which miRNAs were responsible for thede-regulation in insulin expression. In order to narrow down thepossible miRNAs, inventors took a bioinformatics approach integratingresults from the target prediction algorithms TargetScan and Pita aspreviously described [Bartel, D. P. (2009) Cell 136: 215-233; Kertesz M.et al. (2007) Nat. Genet. 39:1278-84] to identify miRNA binding sites(seed-matches) on the 3′UTR of these mRNAs. Inventors found multiplepotential seed matches on the 3′UTR of Sox6 and Bhlhe22 furtherfiltering the list of miRNAs by taking into account only miRNAs whichare expressed in pancreatic islets. Inventors identified several miRNAgenes that appeared to be expressed in islets and potentially repressedeither Sox6 or Bhlhe22 or both (FIG. 6A).

In order to functionally assess potential direct interaction of thecandidate miRNAs with Sox6 and Bhlhe22, inventors cloned their 3′UTRinto vectors expressing a luciferase reporter. These were transfectedseparately into HEK293 cells along with vectors for overexpression ofvarious miRNAs. Inventors then analyzed the relative luciferase activityin the presence of different possible targeting miRNAs compared with anon-targeting miRNA. This analysis revealed that, while a few miRNAs hadno effect, miR-24, miR-129, miR-15/16, miR-26 and miR-27 interacted withthe 3′UTR of Bhlhe22 and repressed the expression of the luciferasereporter by 25-40% (FIG. 6B). Similarly, the 3′UTR of Sox6 was repressedby miR-24, miR182 and miR-375 (FIG. 6C). Interestingly, miR-24interacted with the 3′UTR of both Sox6 and Bhlhe22 and repressed theexpression of the luciferase reporter by

Collectively, our data suggest that miRNAs act to maintain the naturalbalance between transcriptional repressors and activators of the Insulin1 and Insulin 2 genes in adult beta cells. Dicer1 deletion causes animbalance between these transcriptional regulators impacting insulinsynthesis. This in turn compromises glucose homeostasis and promotes therapid onset of diabetes by approximately 40%.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by into thespecification, to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference. In addition, citation oridentification of any reference in this application shall not beconstrued as an admission that such reference is available as prior artto the present invention. To the extent that section headings are used,they should not be construed as necessarily limiting.

1. A method of increasing an insulin content in a pancreatic beta cell,the method comprising expressing in the pancreatic beta cell anexogenous polynucleotide encoding at least one microRNA or a precursorthereof, wherein said microRNA is selected from the group consisting ofmiR-15, miR-16, miR-24, miR-26, miR-27, miR-29, miR-30, miR-129,miR-141, miR-148, miR-182, miR-200, miR-376 and Let-7, therebyincreasing the insulin content in the pancreatic beta cell.
 2. A methodof treating a condition associated with an insulin deficiency in asubject in need thereof, the method comprising administering to thesubject an exogenous polynucleotide encoding at least one microRNA or aprecursor thereof, wherein said microRNA is selected from the groupconsisting of miR-15, miR-16, miR-24, miR-26, miR-27, miR-29, miR-30,miR-129, miR-141, miR-148, miR-182, miR-200, miR-376 and Let-7, therebytreating the condition associated with an insulin deficiency. 3.(canceled)
 4. The method of claim 2, wherein said polynucleotide isoperably linked to a cis acting regulatory element active in pancreaticbeta cell.
 5. The method of claim 2, wherein said condition associatedwith an insulin deficiency comprises diabetes mellitus.
 6. A nucleicacid construct comprising a nucleic acid sequence encoding a microRNA ora precursor thereof said nucleic acid sequence being operably linked toa pancreatic beta cell specific promoter.
 7. The nucleic acid constructof claim 6, wherein said microRNA is selected from the group consistingof miR-15, miR-16, miR-24, miR-26, miR-27, miR-29, miR-30, miR-129,miR-141, miR-148, miR-182, miR-200, miR-376 and Let-7.
 8. Apharmaceutical composition comprising the nucleic acid construct ofclaim 6 and a pharmaceutically acceptable carrier.
 9. An isolatedpancreatic beta cell comprising the nucleic acid construct of claim 6.10. The isolated pancreatic beta cell of claim 9 for the treatment ofdiabetes.
 11. A method of decreasing an insulin content in a pancreaticbeta cell of a subject in need thereof, the method comprisingadministering to the subject an agent capable of downregulatingexpression of at least one microRNA, wherein said microRNA is selectedfrom the group consisting of miR-15, miR-16, miR-24, miR-26, miR-27,miR-29, miR-30, miR-129, miR-141, miR-148, miR-182, miR-200, miR-376 andLet-7, thereby decreasing the insulin content in the pancreatic betacell.
 12. A method of treating a condition associated with elevatedinsulin levels in a subject in need thereof, the method comprisingadministering to the subject an agent capable of downregulatingexpression of at least one microRNA in a pancreatic beta cell of saidsubject, wherein said microRNA is selected from the group consisting ofmiR-15, miR-16, miR-24, miR-26, miR-27, miR-29, miR-30, miR-129,miR-141, miR-148, miR-182, miR-200, miR-376 and Let-7, thereby treatingthe condition associated with elevated insulin levels.
 13. The method ofclaim 11, wherein said agent capable of downregulating expression of atleast one microRNA comprises an enzyme.
 14. The method of claim 13,wherein said enzyme comprises Dicer
 1. 15. The method of claim 13,wherein said enzyme is selected from the group consisting of Drosha,Dicer1, TUT4, DGCR8, exportin 5, Argonaute1, Argonaute2, Argonaute3,Argonaute4, TRBP, smad4 and Ran.
 16. The method of claim 2, wherein saidsubject is a human subject.
 17. A pharmaceutical composition comprisingthe nucleic acid construct of claim 7 and a pharmaceutically acceptablecarrier.
 18. An isolated pancreatic beta cell comprising the nucleicacid construct of claim 7.